Fermentative carotenoid production

ABSTRACT

Novel proteins of microorganism E-396 (FERM BP-4283) and the DNA sequences which encode these proteins have been discovered to provide an improved biosynthetic pathway from farnesyl pyrophosphate and isopentyl pyrophosphate to various carotenoids, especially zeaxanthin, astaxanthin, adonixanthin and canthaxanthin.

This is a divisional of U.S. application Ser. No. 08/980,832, filed Dec.1, 1997, now U.S. Pat. No. 6,291,204.

BACKGROUND OF THE INVENTION

Over 600 different carotenoids have been described from carotenogenicorganisms found among bacteria, yeast, fungi and plants. Currently onlytwo of them, β-carotene and astaxanthin are commercially produced inmicroorganisms and used in the food and feed industry. β-carotene isobtained from algae and astaxanthin is produced in Pfaffia strains whichhave been generated by classical mutation. However, fermentation inPfaffia has the disadvantage of long fermentation cycles and recoveryfrom algae is cumbersome. Therefore it is desirable to developproduction systems which have better industrial applicability, e.g. canbe manipulated for increased titers and/or reduced fermentation times.

Two such systems using the biosynthetic genes form Erwinia herbicola andErwinia uredovora have already been described in WO 91/13078 and EP 393690, respectively. Furthermore, three β-carotene ketolase genes(β-carotene β-4-oxygenase) of the marine bacteria Agrobacteriumaurantiacum and Alcaligenes strain PC-1 (crtW) [Misawa, 1995, Biochem.Biophys. Res. Com. 209 867-876][Misawa, 1995, J. Bacteriology 177,6575-6584 and from the green algae Haematococcus pluvialis (bkt) [Lotan,1995, FEBS Letters 364, 125-128][Kajiwara, 1995, Plant Mol. Biol. 29,343-352] have been cloned. E. coli carrying either the carotenogenicgenes (crtE, crtB, crtY and crtI) of E. herbicola [Hundle, 1994, MGG245, 406-416] or of E. uredovora and complemented with the crtW gene ofA. aurantiacum [Misawa, 1995] or the bkt gene of H. pluvialis [Lotan,1995][Kajiwara, 1995] resulted in the accumulation of canthaxanthin(β,β-carotene-4,4′-dione), originating from the conversion ofβ-carotene, via the intermediate echinenone (β,β-carotene-4-one).

Introduction of the above mentioned genes (crtW or bkt) into E. colicells harbouring besides the carotenoid biosynthesis genes mentionedabove also the crtZ gene of E. uredovora [Kajiwara, 1995][Misawa, 1995],resulted in both cases in the accumulation of astaxanthin(3,3′-dihydroxy-β,β-carotene-4,4′-dione). The results obtained with thebkt gene, are in contrast to the observation made by others [Lotan,1995], who using the same experimental set-up, but introducing the H.pluvialis bkt gene in a zeaxanthin (β,β-carotene-3,3′-diol) synthesisingE. coli host harbouring the carotenoid biosynthesis genes of E.herbicola, a close relative of the above mentioned E. uredovora strain,did not observe astaxanthin production.

SUMMARY OF THE INVENTION

Novel proteins of microorganism E-396 (FERM BP-4283) and the DNAsequences which encode these proteins have been discovered which providean improved biosynthetic pathway from farnesyl pyrophosphate andisopentyl pyrophosphate to various carotenoids, especially zeaxanthin,astaxanthin, adonixanthin and canthaxanthin.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: The biosynthesis pathway for the formation or carotenoids ofFlavobacterium sp. R1534 is illustrated explaining the enzymaticactivities which are encoded by DNA sequences of the present invention.

FIG. 2: Southern blot of genomic Flavobacterium sp. R1534 DNA digestedwith the restriction enzymes shown on top of each lane and hybridizedwith Probe 46F. The arrow indicated the isolated 2.4 kb XhoI/PstIfragment.

FIGS. 3A and B: Southern blot of genomic Flavobacterium sp. R1534 DNAdigested with ClaI or double digested with ClaI and HindIII. Blots shownin Panel A and B were hybridized to probe A or probe B, respectively(see examples). Both ClaI/HindIII fragments of 1.8 kb and 9.2 kb areindicated.

FIG. 4: Southern blot of genomic Flavobacterium sp. R1534 DNA digestedwith the restriction enzymes shown on top of each lane and hybridized toprobe C. The isolated 2.8 kb SalI/HindIII fragment is shown by thearrow.

FIG. 5: Southern blot of genomic Flavobacterium sp. R1534 DNA digestedwith the restriction enzymes shown on top of each lane and hybridized toprobe D. The isolated BclI/SphI fragment of approx. 3 kb is shown by thearrow.

FIG. 6: Physical map of the organization of the carotenoid biosynthesiscluster in Flavobacterium sp. R1534, deduced from the genomic clonesobtained. The location of the probes used for the screening are shown asbars on the respective clones.

FIGS. 7A-7D: Nucleotide sequence of the Flavobacterium sp. R1534carotenoid biosynthesis cluster and its flanking regions (SEQ ID NO: 1).The nucleotide sequence is numbered from the first nucleotide shown (seeBamHI site of FIG. 6). The deduced amino acid sequence of the ORF's(orf-5, orf-1, crtE, crtB, crtI, crtY, crtZ and orf-16) are shown withthe single-letter amino acid code. Arrow (→) indicate the direction ofthe transcription; asterisks, stop codons.

FIG. 8: Protein sequence of the GGPP synthase (crtE) of Flavobacteriumsp. R1534 (SEQ ID NO: 2) with a MW of 31331 Da.

FIG. 9: Protein sequence of the prephytoene synthetase (crtB) ofFlavobacterium sp. R1534 (SEQ ID NO: 3) with a MW of 32615 Da.

FIG. 10: Protein sequence of the phytoene desaturase (crtI) ofFlavobacterium sp. R1534 (SEQ ID NO: 4) with a MW of 54411 Da.

FIG. 11: Protein sequence of the lycopene cyclase (crtY) ofFlavobacterium sp. R1534 (SEQ ID NO: 5) with a MW of 42368 Da.

FIG. 12: Protein sequence of the β-carotene hydroxylase (crtZ) ofFlavobacterium sp. R1534 (SEQ ID NO: 6) with a MW of 19282 Da.

FIG. 13: Recombinant plasmids containing deletions of the Flavobacteriumsp. R1534 carotenoid biosynthesis gene cluster.

FIG. 14: Primers used for PCR reactions (SEQ ID NOs: 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, and 18). The underlined sequence is therecognition site of the indicated restriction enzyme. Small capsindicate nucleotides introduced by mutagenesis. Boxes show theartificial RBS which is recognized in B. subtilis. Small caps in boldshow the location of the original adenine creating the translation startsite (ATG) of the following gene (see original operon). All the ATG's ofthe original Flavobacter carotenoid biosynthetic genes had to bedestroyed to not interfere with the rebuild transcription start site.Arrows indicate start and ends of the indicated Flavobacterium R1534 WTcarotenoid genes.

FIG. 15: Linkers used for the different constructions (SEQ ID NOs: 19,20, 21, 22, 23, 24, 25, and 26). The underlined sequence is therecognition site of the indicated restriction enzyme. Small capsindicate nucleotides introduced by synthetic primers. Boxes show theartificial RBS which is recognized in B. subtilis. Arrow indicate startand ends of the indicated Flavobacterium carotenoid genes.

FIGS. 16A and B: Construction of plasmids pBIIKS(+)-clone59-2, pLyco andpZea4.

FIG. 17: Construction of plasmid p602CAR.

FIG. 18: Construction of plasmids pBIIKS(+)-CARVEG-E and p602 CARVEG-E.

FIGS. 19A-D: Construction of plasmids pHF13-2CARZYIB-EINV andpHP13-2PN25ZYIB-EINV.

FIGS. 20A/D: Construction of plasmid pXI12-ZYIB-EINVMUTRBS2C.

FIGS. 21 and 21B: Northern blot analysis of B. subtilis strainBS1012::ZYIB-EINV4. Panel A: Schematic representation of a reciprocalintegration of plasmid, pXI12-ZYIB-EINV4 into the levan-sucrase gene ofB-subtilis. Panel B: Northern blot obtained with probe A PCR fragmentwhich was obtained with CAR 51 and CAR 76 and hybridizes to the 3′ endof crtZ and the 5′ end or crtY). Panel C: Northern blot obtained withprobe B (BamHI-Xhol fragment isolated from plasmid pBIIKS(+)-crtE/2 andhybridizing to the 5′ part of the crtE gene).

FIG. 22: Schematic representation of the integration sites of threetransformed Bacillus subtilis strains: BS1012::SFCO, BS1012::SFCOCAT1and BA1012::SFCONEO1. Amplification of the synthetic Flavobacteriumcarotenoid operon (SFCO) can only be obtained in those strains havingamplifiable structures. Probe A was used to determine the copy number ofthe integrated SFCO. Erythromycine resistance gene (ermAM),chloramphenicol resistance gene (cat), neomycine resistance gene (neo),terminator of the cryT gene of B. subtilis (cryT), levan-sucrase gene(sac-B 5′ and sac-B 3′), plasmid sequences of pXI12 (pXI12), promoteroriginating from site I of the veg promoter complex (PvegI).

FIGS. 23A-C: Construction of plasmids pXI12-ZYIB-EINV4MUTRBS2CNEO andpXI12-ZYIB-EINV4MUTRBS2CCAT.

FIGS. 24A-L: Complete nucleotide sequence of plasmid pZea4 (SEQ ID NO:27).

FIGS. 25A and 25B: Synthetic crtW gene of Alcaligenes PC-1 (SEQ ID NO:28). The translated protein sequence (SEQ ID NO: 29) is shown above thedouble stranded DNA sequence. The twelve oligonucleotides (crtW1-crtW12)used for the PCR synthesis are underlined.

FIG. 26: Construction of plasmid pBIIKS-crtEBIYZW. The HindIII-Pm1Ifragment of pALTER-Ex2-crtW, carrying the synthetic crtW gene, wascloned into the HindIII and MluI (blunt) sites. PvegI and Ptac are thepromoters used for the transcription of the two opera. The CoIE1replication origin of this plasmid is compatible with the p15A originpresent in the pALTER-Ex2 constructs.

FIG. 27: Relevant inserts of all plasmids constructed in Example 7.Disrupted genes are shown by //. Restriction sites: S=SacI, X=XbaI,H=HindIII, N=NsiI, Hp=HpaI, Nd=NdeI.

FIG. 28: Reaction products (carotenoids) obtained from β-carotene by theprocess of the present invention.

FIG. 29: Isolation of the crt cluster of the strain E-396. Genomic DNAof E-396 was digested overnight with different combinations ofrestrictions enzymes and separated by agarose gel electrophoresis beforetransferring the resulting fragments by Southern blotting onto anitrocellulose membrane. The blot was hybridised with a ³²P labelled 334bp fragment obtained by digesting the aforementioned PCR fragmentJAPclone8 with BssHII and MluI. An approx. 9,4 kb EcoRI/BamHI fragmenthybridizing to the probe was identified as the most appropiate forcloning since it is long enough to potentially carry the complete crtcluster. The fragment was isolated and cloned into the EcoRI and BamHIsites of pBluescriptIIKS resulting in plasmid pJAPCL544.

FIGS. 30A-30B Shows the sequence obtained containing the crtW_(E396)(from nucleotide 40 to 768) and crtZ_(E396) (from nucleotide 765 to1253) genes of the bacterium E-396 (SEQ ID NO: 30).

FIG. 31: The nucleotide sequence of the crtW_(E396) gene (SEQ ID NO:31).

FIG. 32: The amino acid sequence encoded by the crtW_(E396) (SEQ ID NO:32) gene shown in FIG. 31.

FIG. 33: The nucleotide sequence of the crtZ_(E396) (SEQ ID NO: 33)gene.

FIG. 34: The amino acid sequence (SEQ ID NO: 34) encoded by thecrtZ_(E396) gene shown in FIG. 33.

FIG. 35: Diagram of plasmid pUC18-E396crtWZPCR.

FIG. 36: Construction of plasmid pBIIKS-crtEBIY[E396WZ].

FIG. 37: Construction of plasmid pBIIKS-crtEBIY[E396W]DZ which has atruncated non-functional crtZ gene.

FIGS. 38A and 39B: 463 bp PstI-BamHI fragment (SEQ ID NO: 35)originating from the 3′ end of the insert of pJAPCL544 (FIG. 29)highlighted a ˜1300 bp-long PstI-PstI fragment. This fragment wasisolated and cloned into the PstI site of pBSIIKS(+) resulting inplasmid pBSIIKS-#1296. The sequence of the insert is shown (small capletters refer to new sequence obtained. Capital letters show thesequence also present in the 3′ of the insert of plasmid pJAPCL544).

FIGS. 39A and 39B: The DNA sequence of the complete crtE_(E396) gene(SEQ ID NO: 36).

FIG. 40: The amino acid sequence encoded by the crtE_(E396) gene (SEQ IDNO: 37) shown in FIG. 39 (SEQ ID NO: 36).

FIG. 41: Construction of plasmid carrying the complete crt cluster ofE-396 (pE396CARcrtW-E).

FIG. 42: Construction of plasmid pRSF1010-Amp^(r).

FIG. 43: Construction of plasmids RSF1010-Amp^(r)-crt1 andRSF1010-Ampr-crt2.

DETAILED DESCRIPTION OF THE INVENTION

Novel proteins of microorganism E-396 (FERM BP-4283) and the DNAsequences which encode these proteins have been discovered which providean improved biosynthetic pathway from farnesyl pyrophosphate andisopentyl pyrophosphate to various carotenoids, especially zeaxanthin,astaxanthin, adonixanthin and canthaxanthin.

One aspect of the invention is a polynucleotide comprising a DNAsequence which encodes the GGPP synthase (crtE_(E396)) (SEQ ID NO: 37)of microorganism E-396, said polynucleotide being substantially free ofother polynucleotides of microorganism E-396. Also encompassed by thisaspect of the present invention is a polynucleotide comprising a DNAsequence which is substantially homologous to said DNA sequence. SaidGGPP synthase catalyzes the condensation of farnesyl pyrophosphate andisopentyl pyrophosphate to obtain geranylgeranyl pyrophosphate, acarotenoid precursor. The preferred GGPP synthase has the amino acidsequence of FIG. 40 (SEQ ID NO: 37), and the preferred DNA sequenceencodes said amino acid sequence. The especially preferred DNA sequenceis shown in FIG. 39 (SEQ ID NO: 36).

This aspect of the present invention also includes a vector comprisingthe aforesaid polynucleotide, preferably in the form of an expressionvector. Furthermore this aspect of the present invention also includes arecombinant cell comprising a host cell which is transformed by theaforesaid polynucleotide or vector which contains such a polynucleotide.Preferably said host cell is a prokaryotic cell and more preferably saidhost cell is E. coli or a Bacillus strain. However, said host cell mayalso be a eukaryotic cell, preferably a yeast cell or a fungal cell.

Finally this aspect of the present invention also comprises a processfor the preparation of geranylgeranyl pyrophosphate by culturing saidrecombinant cell of the invention containing farnesyl pyrophosphate andisopentyl pyrophosphate in a culture medium under suitable cultureconditions whereby said GGPP synthase is expressed by said cell andcatalyzes the condensation of farnesyl pyrophosphate and isopentylpyrophosphate to geranylgeranyl pyrophosphate, and isolating thegeranylgeranyl pyrophosphate from such cells or the culture medium.

Another aspect of the present invention is a polynucleotide comprising aDNA sequence which encodes said β-carotene hydroxylase of microorganismE-396 (crtZ_(E396)) (SEQ ID NO: 34), said polynucleotide beingsubstantially free of other polynucleotides of microorganism E-396. Alsoencompassed by this aspect of the present invention is a polynucleotidecomprising a DNA sequence which is substantially homologous to said DNAsequence. Said β-carotene hydroxylase catalyzes the hydroxylation ofβ-carotene to produce the xanthophyll, zeaxanthin. The preferredβ-carotene hydroxylase has the amino acid sequence of FIG. 34 (SEQ IDNO: 34), and the preferred DNA sequence is one which encodes said aminoacid sequence. The especially preferred DNA sequence is a DNA sequencecomprising the sequence shown in FIG. 33 (SEQ ID NO: 33).

This aspect of the present invention also includes a vector comprisingthe aforesaid polynucleotide, preferably in the form of an expressionvector. Furthermore this aspect of the present invention also includes arecombinant cell comprising a host cell which is transformed by theaforesaid polynucleotide or vector which contains such a DNA sequence.Preferably said host cell is a prokaryotic cell and more preferably saidhost cell is E. coli or a Bacillus strain. However, said host cell mayalso be a eukaryotic cell, preferably a yeast cell or a fungal cell.

Finally this aspect of the present invention also comprises a processfor the preparation of zeaxanthin by culturing said recombinant cell ofthe invention containing β-carotene in a culture medium under suitableculture conditions whereby said β-carotene hydroxylase is expressed bysaid cell and catalyzes the hydroxylation of β-carotene to produce thexanthophyll, zeaxanthin, and isolating the zeaxanthin from such cells orthe culture medium.

Another aspect of the present invention is a polynucleotide comprising aDNA sequence which encodes said β-carotene hydroxylase of microorganismE-396 (crtW_(E396)) (SEQ ID NO: 32), said polynucleotide beingsubstantially free of other polynucleotides of microorganism E-396. Alsoencompassed by this aspect of the present invention is a polynucleotidecomprising a DNA sequence which is substantially homologous to said DNAsequence. Said β-carotene β4-oxygenase catalyzes the hydroxylation ofβ-carotene to produce the echinenone, and, with the further catalysis ofechinenone by the enzyme encoded by crtW_(E396), to canthaxanthin. Thepreferred β-carotene β4-oxygenase has the amino acid sequence of FIG. 32(SEQ ID NO: 32), and the preferred DNA sequence is one which encodessaid amino acid sequence. The especially preferred DNA sequence is a DNAsequence comprising the sequence shown in FIG. 31 (SEQ ID NO: 31).

This aspect of the present invention also includes a vector comprisingthe aforesaid polynucleotide, preferably in the form of an expressionvector. Furthermore this aspect of the present invention also includes arecombinant cell comprising a host cell which is transformed by theaforesaid polynucleotide or vector which contains such a DNA sequence.Preferably said host cell is a prokaryotic cell and more preferably saidhost cell is E. coli or a Bacillus strain. However, said host cell mayalso be a eukaryotic cell, preferably a yeast cell or a fungal cell.

Finally this aspect of the present invention also comprises a processfor the preparation of canthaxanthin by culturing said recombinant cellof the invention containing β-carotene in a culture medium undersuitable culture conditions whereby said β-carotene β4-oxygenase isexpressed by said cell and catalyzes the conversion of β-carotene toproduce echinenone and through further catalysis to producecanthaxanthin, and isolating the canthaxanthin from such cells or theculture medium.

It is contemplated, and in fact preferred, that the aforementioned DNAsequences, crtE_(E396), crtW_(E396) and crtZ_(E396), which terms referto the above-described genes of microorganism E-396 encompassed by theinvention herein described, are incorporated, especially crtW_(E396) andcrtZ_(E396), with selected DNA sequences from Flavobacterium sp. R1534into a polynucleotide of the invention whereby two or more of said DNAsequences which encode enzymes catalyzing contiguious steps in theprocess shown in FIGS. 1 and 28 are contained in said polynucleotide,said polynucleotide being substantially free of other polynucleotides ofmicroorganism E-396 and Flavobacterium sp. R1534, to obtain advantageousproduction of the carotenoids canthaxanthin, zeaxanthin, astaxanthin andadonixanthin.

Thus, one embodiment of the present invention is a process for thepreparation of zeaxanthin which process comprises culturing arecombinant cell containing farnesyl pyrophosphate and isopentylpyrophosphate under culture conditions sufficient for the expression ofenzymes which catalyze the conversion of the farnesyl pyrophosphate andisopentyl pyrophosphate to zeaxanthin, said recombinant cell comprisinga host cell transformed by an expression vector comprising a regulatorysequence and a polynucleotide containing DNA sequences which encode saidenzymes, as follows:

a) a DNA sequence which encodes the GGPP synthase of Flavobacterium sp.R1534 (crtE) (SEQ ID NO: 2) or a DNA sequence which is substantiallyhomologous,

b) a DNA sequence which encodes the prephytoene synthase ofFlavobacterium sp. R1534 (crtB) (SEQ ID NO: 3) or a DNA sequence whichis substantially homologous,

c) a DNA sequence which encodes the phytoene desaturase ofFlavobacterium sp. R1534 (crtI) (SEQ ID NO: 4) or a DNA sequence whichis substantially homologous,

d) a DNA sequence which encodes the lycopene cyclase of Flavobacteriumsp. R1534 (crtY) (SEQ ID NO: 5) or a DNA sequence which is substantiallyhomologous,

e) a DNA sequence which encodes the β-carotene hydroxylase ofmicroorganism E-396 (crtZ_(E396)) (SEQ ID NO: 34) or a DNA sequencewhich is substantially homologous; and isolating the zeaxanthin fromsuch cells or the culture medium.

The above-described polynucleotide encodes enzymes which catalyze theconversion of farnesyl pyrophosphate and isopentyl pyrophosphate tozeaxanthin. It is preferred that this embodiment of the inventionutilize a polynucleotide containing crtE, crtB, crtI, crtY, andcrtZ_(E396).

It is especially preferred that for this embodiment of the invention:

a) the GGPP synthase has the amino acid sequence of FIG. 8 (SEQ ID NO:2),

b) the prephytoene synthase has the amino acid sequence of FIG. 9 (SEQID NO: 3),

c) the phytoene desaturase has the amino acid sequence of FIG. 10 (SEQID NO: 4),

d) the lycopene cyclase has the amino acid sequence of FIG. 11 (SEQ IDNO: 5), and

e) the β-carotene hydroxylase has the amino acid sequence of FIG. 34.

It is most preferred that for this embodiment of the invention:

a) the DNA sequence encoding the GGPP synthase comprises bases 2521-3408of FIG. 7 (SEQ ID NO: 1),

b) the DNA sequence encoding the prephytoene synthase comprises bases4316-3405 of FIG. 7 (SEQ ID NO: 1),

c) the DNA sequence encoding the phytoene desaturase comprises bases4313-5797 of FIG. 7 (SEQ ID NO: 1),

d) the DNA sequence encoding the lycopene cyclase comprises bases5794-6942 of FIG. 7 (SEQ ID NO: 1), and

e) the DNA sequence encoding the β-carotene hydroxylase comprises thesequence of FIG. 33 (SEQ ID NO: 33).

A second embodiment of the invention is a process for the preparation ofcanthaxanthin which process comprises culturing a recombinant cellcontaining farnesyl pyrophosphate and isopentyl pyrophosphate underculture conditions sufficient for the expression of enzymes whichcatalyze the conversion of the farnesyl pyrophosphate and isopentylpyrophosphate to canthaxanthin, said recombinant cell comprising a hostcell transformed by an expression vector comprising a regulatorysequence and a polynucleotide containing DNA sequences which encode saidenzymes, as follows:

a) a DNA sequence which encodes the GGPP synthase of Flavobacterium sp.R1534 (crtE) (SEQ ID NO: 2) or a DNA sequence which is substantiallyhomologous,

b) a DNA sequence which encodes the prephytoene synthase ofFlavobacterium sp. R1534 (crtB) (SEQ ID NO: 3) or a DNA sequence whichis substantially homologous,

c) a DNA sequence which encodes the phytoene desaturase ofFlavobacterium sp. R1534 (crtI) (SEQ ID NO: 4) or a DNA sequence whichis substantially homologous,

d) a DNA sequence which encodes the lycopene cyclase of Flavobacteriumsp. R1534 (crtY) (SEQ ID NO: 5) or a DNA sequence which is substantiallyhomologous, and

e) a DNA sequence which encodes the β-carotene β4-oxygenase ofmicroorganism E-396 (crtW_(E396)) (SEQ ID NO: 32) or a DNA sequencewhich is substantially homologous; and isolating the canthaxanthin fromsuch cells or the culture medium.

The above-described polynucleotide encodes enzymes which catalyze theconversion of farnesyl pyrophosphate and isopentyl pyrophosphate tocanthaxanthin. It is preferred that this embodiment of the inventionutilize a polynucleotide containing crtE, crtB, crtI, crtY, andcrtW_(E396).

It is especially preferred that for this embodiment of the invention:

a) the GGPP synthase has the amino acid sequence of FIG. 8 (SEQ ID NO:2),

b) the prephytoene synthase has the amino acid sequence of FIG. 9 (SEQID NO: 3),

c) the phytoene desaturase has the amino acid sequence of FIG. 10 (SEQID NO: 4),

d) the lycopene cyclase has the amino acid sequence of FIG. 11 (SEQ IDNO: 5), and

e) the β-carotene β4-oxygenase has the amino acid sequence of FIG. 32(SEQ ID NO: 32).

For this embodiment of the invention, it is most preferred that:

a) the DNA sequence encoding the GGPP synthase comprises bases 2521-3408of FIG. 7 (SEQ ID NO: 1),

b) the DNA sequence encoding the prephytoene synthase comprises bases4316-3405 of FIG. 7 (SEQ ID NO: 1),

c) the DNA sequence encoding the phytoene desaturase comprises bases4313-5797 of FIG. 7 (SEQ ID NO: 1),

d) the DNA sequence encoding the lycopene cyclase comprises bases5794-6942 of FIG. 7 (SEQ ID NO: 1), and

e) the DNA sequence encoding the β-carotene β4-oxygenase comprises thesequence of FIG. 31.

A third embodiment of the invention is a process for the preparation ofastaxanthin and adonixanthin wherein said process comprises culturing arecombinant cell containing farnesyl pyrophosphate and isopentylpyrophosphate under culture conditions sufficient for the expression ofenzymes which catalyze the conversion of the farnesyl pyrophosphate andisopentyl pyrophosphate to astaxanthin and adonixanthin, saidrecombinant cell comprising a host cell transformed by an expressionvector comprising a regulatory sequence and a polynucleotide containingDNA sequences which encode said enzymes, as follows:

a) a DNA sequence which encodes the GGPP synthase of Flavobacterium sp.R1534 (crtE) (SEQ ID NO: 2) or a DNA sequence which is substantiallyhomologous,

b) a DNA sequence which encodes the prephytoene synthase ofFlavobacterium sp. R1534 (crtB) (SEQ ID NO: 3) or a DNA sequence whichis substantially homologous,

c) a DNA sequence which encodes the phytoene desaturase ofFlavobacterium sp. R1534 (crtI) (SEQ ID NO: 4) or a DNA sequence whichis substantially homologous,

d) a DNA sequence which encodes the lycopene cyclase of Flavobacteriumsp. R1534 (crtY) (SEQ ID NO: 5) or a DNA sequence which is substantiallyhomologous,

e) a DNA sequence which encodes the β-carotene b4-oxygenase ofFlavobacterium sp. R1534 (crtW) or a DNA sequence which is substantiallyhomologous, and

f) a DNA sequence which encodes the β-carotene hydroxylase ofmicroorganism E-396 (crtZ_(E396)) or a DNA sequence which issubstantially homologous; and isolating the astaxanthin and adonixanthinfrom such cells or the culture medium.

The above-described polynucleotide encodes enzymes which catalyze theconversion of farnesyl pyrophosphate and isopentyl pyrophosphate toastaxanthin and adonixanthin. It is preferred that this embodiment ofthe invention utilize a polynucleotide containing crtE, crtB, crtI,crtY, crtW, and crtZ_(E396) (SEQ ID NO: 34).

It is especially preferred that for this embodiment of the invention:

a) the GGPP synthase has the amino acid sequence of FIG. 8 (SEQ ID NO:2),

b) the prephytoene synthase has the amino acid sequence of FIG. 9 (SEQID NO: 3),

c) the phytoene desaturase has the amino acid sequence of FIG. 10 (SEQID NO: 4),

d) the lycopene cyclase has the amino acid sequence of FIG. 11 (SEQ IDNO: 5),

e) the β-carotene β4-oxygenase has the amino acid sequence of FIG. 25(SEQ ID NO: 29), and

f) the β-carotene hydroxylase has the amino acid sequence of FIG. 34(SEQ ID NO: 34).

It is most preferred that for this embodiment of the invention:

a) the DNA sequence encoding the GGPP synthase comprises bases 2521-3408of FIG. 7 (SEQ ID NO: 1),

b) the DNA sequence encoding the prephytoene synthase comprises bases4316-3405 of FIG. 7 (SEQ ID NO: 1),

c) the DNA sequence encoding the phytoene desaturase comprises bases4313-5797 of FIG. 7 (SEQ ID NO: 1),

d) the DNA sequence encoding the lycopene cyclase comprises bases5794-6942 of FIG. 7 (SEQ ID NO: 1),

e) the DNA sequence encoding the β-carotene β4-oxygenase comprises thesequence of FIG. 25 (SEQ ID NO: 28), and

f) the DNA sequence encoding the β-carotene hydroxylase comprises thesequence of FIG. 33 (SEQ ID NO: 33).

A fourth embodiment of the invention is a process for the preparation ofastaxanthin and adonixanthin wherein said process comprises culturing arecombinant cell containing farnesyl pyrophosphate and isopentylpyrophosphate under culture conditions sufficient for the expression ofenzymes which catalyze the conversion of the farnesyl pyrophosphate andisopentyl pyrophosphate to astaxanthin and adonixanthin, saidrecombinant cell comprising a host cell transformed by an expressionvector comprising a regulatory sequence and a polynucleotide containingDNA sequences which encode said enzymes, as follows:

a) a DNA sequence which encodes the GGPP synthase of Flavobacterium sp.R1534 (crtE) (SEQ ID NO: 2) or a DNA sequence which is substantiallyhomologous,

b) a DNA sequence which encodes the prephytoene synthase ofFlavobacterium sp. R1534 (crtB) (SEQ ID NO: 3) or a DNA sequence whichis substantially homologous,

c) a DNA sequence which encodes the phytoene desaturase ofFlavobacterium sp. R1534 (crtI) (SEQ ID NO: 4) or a DNA sequence whichis substantially homologous,

d) a DNA sequence which encodes the lycopene cyclase of Flavobacteriumsp. R1534 (crtY) (SEQ ID NO: 5) or a DNA sequence which is substantiallyhomologous,

e) a DNA sequence which encodes the β-carotene β4-oxygenase ofmicroorganism E-396 (crtW_(E396)) (SEQ ID NO: 32) or a DNA sequencewhich is substantially homologous, and

f) a DNA sequence which encodes the β-carotene hydroxylase ofmicroorganism E-396 (crtZ_(E396)) (SEQ ID NO: 34) or a DNA sequencewhich is substantially homologous; and isolating the astaxanthin andadonixanthin from such cells or the culture medium.

The above-described polynucleotide encodes enzymes which catalyze theconversion of farnesyl pyrophosphate and isopentyl pyrophosphate toastaxanthin and adonixanthin. It is preferred that this embodiment ofthe invention utilize a polynucleotide containing crtE, crtB, crtI,crtY, crtW_(E396), and crtZ_(E396).

It is especially preferred that for this embodiment of the invention:

a) the GGPP synthase has the amino acid sequence of FIG. 8 (SEQ ID NO:2),

b) the prephytoene synthase has the amino acid sequence of FIG. 9 (SEQID NO: 3),

c) the phytoene desaturase has the amino acid sequence of FIG. 10 (SEQID NO: 4),

d) the lycopene cyclase has the amino acid sequence of FIG. 11 (SEQ IDNO: 5),

e) the β-carotene β4oxygenase has the amino acid sequence of FIG. 32(SEQ ID NO: 32), and

f) the β-carotene hydroxylase has the amino acid sequence of FIG. 34(SEQ ID NO: 34).

It is most preferred that for this embodiment of the invention:

a) the DNA sequence encoding the GGPP synthase comprises bases 2521-3408of FIG. 7 (SEQ ID NO: 1),

b) the DNA sequence encoding the prephytoene synthase comprises bases4316-3405 of FIG. 7 (SEQ ID NO: 1),

c) the DNA sequence encoding the phytoene desaturase comprises bases4313-5797 of FIG. 7 (SEQ ID NO: 1),

d) the DNA sequence encoding the lycopene cyclase comprises bases5794-6942 of FIG. 7 (SEQ ID NO: 1),

e) the DNA sequence encoding the β-carotene β4-oxygenase comprises thesequence of FIG. 31 (SEQ ID NO: 31), and

f) the DNA sequence encoding the E-carotene hydroxylase comprises thesequence of FIG. 33 (SEQ ID NO: 33).

A fifth embodiment of the present invention is a process for thepreparation of adonixanthin wherein said process comprises culturing arecombinant cell containing farnesyl pyrophosphate and isopentylpyrophosphate under culture conditions sufficient for the expression ofenzymes which catalyze the conversion of the farnesyl pyrophosphate andisopentyl pyrophosphate to adonixanthin, said recombinant cellcomprising a host cell transformed by an expression vector comprising aregulatory sequence and a polynucleotide containing DNA sequences whichencode said enzymes, as follows:

a) a DNA sequence which encodes the GGPP synthase of microorganism E-396(crtE_(E396)) (SEQ ID NO: 37) or a DNA sequence which is substantiallyhomologous,

b) a DNA sequence which encodes the prephytoene synthase ofmicroorganism E-396 (crtB_(E396)) or a DNA sequence which issubstantially homologous,

c) a DNA sequence which encodes the phytoene desaturase of microorganismE-396 (crtI_(E396)) or a DNA sequence which is substantially homologous,

d) a DNA sequence which encodes the lycopene cyclase of microorganismE-396 (crtY_(E396)) or a DNA sequence which is substantially homologous,

e) a DNA sequence which encodes the b-carotene b4-oxygenase ofmicroorganism E-396 (crtW_(E396)) (SEQ ID NO: 32) or a DNA sequencewhich is substantially homologous, and

f) a DNA sequence which encodes the β-carotene hydroxylase ofmicroorganism E-396 (crtZ_(E396)) (SEQ ID NO: 33) or a DNA sequencewhich is substantially homologous,

said host cell being substantially free of other polynucleotides ofmicroorganism E-396; and isolating the adonixanthin from such cells orthe culture medium.

The above-described polynucleotide encodes enzymes which catalyze theconversion of farnesyl pyrophosphate and isopentyl pyrophosphate toadonixanthin. It is preferred that this embodiment of the inventionutilize a polynucleotide containing crtE_(E396), crtB_(E396),crtI_(E396), crtY_(E396), crtW_(E396), and crtZ_(E396). It has beenfound that the use of the above-described process of the inventionresults in a preferential production of adonixanthin in relation toastaxanthin and other carotenoids. The preferred polynucleotide isplasmid pE396CARcrtW-E whose construction is described in Example 9herein.

The present invention also comprises the polynucleotides described abovefor the various embodiments of the invention and a vector comprisingsuch a polynucleotide, preferably in the form of an expression vector.Furthermore the present invention also comprises a recombinant cellwherein said cell is a host cell which is transformed by apolynucleotide of the invention or vector which contains such apolynucleotide. Host cells useful for the expression of heterologousgenes normally contain farnesyl pyrophosphate and isopentylpyrophosphate, which are used for other purposes within the cell.Preferably said host cell is a prokaryotic cell and more preferably saidhost cell is an E. coli or a Bacillus strain. However, said host cellmay also be a eukaryotic cell, preferably a yeast cell or a fungal cell.

Finally the present invention also comprises a process for thepreparation of a desired carotenoid by culturing a recombinant cell ofthe invention containing a starting material in a culture medium undersuitable culture conditions and isolating the desired carotenoid fromsuch cells or the culture medium wherein the cell utilizes thepolynucleotide of the invention which contains said DNA sequences toexpress the enzymes which catalyze the reactions necessary to producethe desired carotenoid from the starting material. Where an enzymecatalyzes two sequential steps and it is preferred to produce theproduct of the second step (such as producing astaxanthin preferentiallyto adonixanthin (see FIG. 28)), a higher copy number of the DNA sequenceencoding the enzyme may be used to further production of the product ofthe second of the two steps in comparison to the first product. Thepresent invention further comprises a process for the preparation of afood or feed composition which process comprises mixing a nutritionallyeffective amount of the carotenoid isolated from the aforementionedrecombinant cells or culture medium with said food or feed.

In this context it should be mentioned that the expression “a DNAsequence is substantially homologous” refers with respect to the crtEencoding DNA sequence to a DNA sequence which encodes an amino acidsequence which shows more than 45%, preferably more than 60% and morepreferably more than 75% and most preferably more than 90% identicalamino acids when compared to the amino acid sequence of crtE ofFlavobacterium sp. 1534 and is the amino acid sequence of a polypeptidewhich shows the same type of enzymatic activity as the enzyme encoded bycrtE of Flavobacterium sp. 1534. In analogy with respect to crtB thismeans more than 60%, preferably more than 70%, more preferably more than80% and most preferably more than 90%; with respect to crtI this meansmore than 70%, preferably more than 80% and most preferably more than90%; with respect to crtY this means 55%, preferably 70%, morepreferably 80% and most preferably 90%.

“DNA sequences which are substantially homologous” refer with respect tothe crtW_(E396) encoding DNA sequence to a DNA sequence which encodes anamino acid sequence which shows more than 60%, preferably more than 75%and most preferably more than 90% identical amino acids when compared tothe amino acid sequence of crtW of the microorganism E 396 (FERMBP-4283) and is the amino acid sequence of a polypeptide which shows thesame type of enzymatic activity as the enzyme encoded by crtW of themicroorganism E 396. In analogy with respect to crtZ_(E396) this meansmore than 75%, preferable more than 80% and most preferably more than90%; with respect to crtE_(E396), crtB_(E396), crtI_(E396), crtY_(E396)and crtZ_(E396) this means more than 80%, preferably more than 90% andmost preferably 95%.

The expression “said polynucleotide being substantially free of otherpolynucleotides of Flavobacterium sp. R1534” and “said polynucleotidebeing substantially free of other polynucleotides of microorganismE-396” is meant to preclude the present invention from encompassing thepolynucleotides as they exist in Flavobacterium sp. R1534 or inmicroorganism E-396, themselves. The polynucleotides herein describedwhich are combinations of two or more DNA sequences of Flavobacteriumsp. R1534 and/or microorganism E-396 are also substantially free ofother polynucleotides of Flavobacterium sp. R1534 and microorganismE-396 in any circumstance where a polynucleotide containing only asingle such DNA sequence would be substantially free of otherpolynucleotides of Flavobacterium sp. R1534 or microorganism E-396.

DNA sequences in form of genomic DNA, cDNA or synthetic DNA can beprepared as known in the art [see e.g. Sambrook et al., MolecularCloning, Cold Spring Habor Laboratory Press 1989] or, e.g. asspecifically described in Examples 1, 2 or 7. In the context of thepresent invention it should be noted that all DNA sequences used for theprocess for production of carotenoids of the present invention encodingcrt-gene products can also be prepared as synthetic DNA sequencesaccording to known methods or in analogy to the method specificallydescribed for crtW in Example 7.

The cloning of the DNA-sequences of the present invention from suchgenomic DNA can than be effected, e.g. by using the well knownpolymerase chain reaction (PCR) method. The principles of this methodare outlined e.g. in PCR Protocols: A guide to Methods and Applications,Academic Press, Inc. (1990). PCR is an in vitro method for producinglarge amounts of a specific DNA of defined length and sequence from amixture of different DNA-sequences. Thereby, PCR is based on theenzymatic amplification of the specific DNA fragment of interest whichis flanked by two oligonucleotide primers which are specific for thissequence and which hybridize to the opposite strand of the targetsequence. The primers are oriented with their 3′ ends pointing towardeach other. Repeated cycles of heat denaturation of the template,annealing of the primers to their complementary sequences and extensionof the annealed primers with a DNA polymerase result in theamplification of the segment between the PCR primers. Since theextension product of each primer can serve as a template for the other,each cycle essentially doubles the amount of the DNA fragment producedin the previous cycle.

By utilizing the thermostable Taq DNA polymerase, isolated from thethermophilic bacteria Thermus aquaticus, it has been possible to avoiddenaturation of the polymerase which necessitated the addition of enzymeafter each heat denaturation step. This development has led to theautomation of PCR by a variety of simple temperature-cycling devices. Inaddition, the specificity of the amplification reaction is increased byallowing the use of higher temperatures for primer annealing andextension. The increased specificity improves the overall yield ofamplified products by minimizing the competition by non-target fragmentsfor enzyme and primers. In this way the specific sequence of interest ishighly amplified and can be easily separated from the non-specificsequences by methods known in the art, e.g. by separation on an agarosegel and cloned by methods known in the art using vectors as describede.g. by Holten and Graham in Nucleic Acid Res. 19, 1156 (1991), Kovalicet. al. in Nucleic Acid Res. 19, 4560 (1991), Marchuk et al. in NucleicAcid Res. 19, 1154 (1991) or Mead et al. in Bio/Technology 9,657-663(1991).

The oligonucleotide primers used in the PCR procedure can be prepared asknown in the art and described e.g. in Sambrook et al., s.a.

Amplified DNA-sequences can than be used to screen DNA libraries bymethods known in the art (Sambrook et al., s.a.) or as specificallydescribed in Examples 1 and 2.

Once complete DNA-sequences of the present invention have been obtainedthey can be used as a guideline to define new PCR primers for thecloning of substantially homologous DNA sequences from other sources. Inaddition they and such homologous DNA sequences can be integrated intovectors by methods known in the art and described, e.g., in Sambrook etal. (s.a.) to express or overexpress the encoded polypeptide(s) inappropriate host systems. The expression vector into which thepolynucleotides of the invention are integrated is not critical.Conventional expression vectors may be selected based upon the size ofthe polynucleotide of the invention to be inserted into the vector andthe host cell to be transformed by the vector. Such conventionalexpression vectors contain a regulatory sequence for the synthesis ofmRNA derived from the polynucleotide of the invention being expressedand possible marker genes. Conventional regulatory sequences generallycontain, but are not limited to, one or more of the following: a signalsequence, an origin of replication, one or more marker genes, anenhancer element, a promoter, and a transcription termination sequence.

However, a man skilled in the art knows that also the DNA-sequencesthemselves can be used to transform the suitable host systems of theinvention to get overexpression of the encoded polypeptide. Appropriatehost systems are for example Bacteria e.g. E. coli, Bacilli as, e.g.Bacillus subtilis or Flavobacter strains. E. coli, which could be usedare E. coli K12 strains e.g. M15 [described as DZ 291 by Villarejo etal. in J. Bacteriol. 120, 466-474 (1974)], HB 101 [ATCC No. 33694] or E.coli SG13009 [Gottesman et al., J. Bacteriol. 148, 265-273 (1981)].

Suitable Flavobacter strains can be obtained from any of the culturecollections known to the man skilled in the art and listed, e.g. in thejournal “Industrial Property” (January 1994, pgs 29-40), like theAmerican Type Culture Collection (ATCC) or the Centralbureau voorSchimmelkultures (CBS) and are, e.g. Flavobacterium sp. R 1534 (ATCC No.21588, classified as unknown bacterium; or as CBS 519.67) or allFlavobacter strains listed as CBS 517.67 to CBS 521.67 and CBS 523.67 toCBS 525.67, especially R 1533 (which is CBS 523.67 or ATCC 21081,classified as unknown bacterium; see also U.S. Pat. No. 3,841,967).Further Flavobacter strains are also described in WO 91/03571. Suitableeukaryotic host systems are for example fungi, like Aspergilli, e.g.Aspergillus niger [ATCC 9142] or yeasts, like Saccharomyces, e.g.Saccharomyces cerevisiae or Pichia, like pastoris, all available fromATCC.

Suitable vectors which can be used for expression in E. coli arementioned, e.g., by Sambrook et al. [s.a.] or by Fiers et al. in Procd.8th Int. Biotechnology Symposium” [Soc. Franc. de Microbiol., Paris(Durand et al., eds.), pp. 680-697 (1988)] or by Bujard et al. inMethods in Enzymology, eds. Wu and Grossmann, Academic Press, Inc. Vol.155, 416-433 (1987) and Stuber et al. in Immunological Methods, eds.Lefkovits and Pernis, Academic Press, Inc., Vol. IV, 121-152 (1990).Vectors which could be used for expression in Bacilli are known in theart and described, e.g., in EP 405 370, EP 635 572 Procd. Nat. Acad.Sci. USA 81, 439 (1984) by Yansura and Henner, Meth. Enzym. 185, 199-228(1990) or EP 207 459. Vectors which can be used for expression in fungiare known in the art and described e.g. in EP 420 358 and for yeast inEP 183 070, EP 183 071, EP 248 227, EP 263 311. Vectors which can beused for expression in Flavobacter are known in the art and described inthe Examples or, e.g. in Plasmid Technology, edt. by J. Grinsted and P.M. Bennett, Academic Press (1990).

Once such DNA-sequences have been expressed in an appropriate host cellin a suitable medium, the carotenoids can be isolated either from themedium in the case they are secreted into the medium or from the hostorganism and, if necessary separated from other carotenoids if presentin case one specific carotenoid is desired by methods known in the art(see e.g. Carotenoids Vol IA: Isolation and Analysis, G. Britton, S.Liaaen-Jensen, H. Pfander; 1995, Birkhäuser Verlag, Basel).

The carotenoids of the present invention can be used in a process forthe preparation of food or feeds. A man skilled in the art is familiarwith such processes. Such compound foods or feeds can further compriseadditives or components generally used for such purpose and known in thestate of the art.

After the invention has been described in general hereinbefore, thefollowing examples are intended to illustrate details of the invention,without thereby limiting it in any matter.

EXAMPLE 1

Materials and General Methods Used

Bacterial strains and plasmids: Flavobacterium sp. R1534 WT (ATCC 21588)was the DNA source for the genes cloned. Partial genomic libraries ofFlavobacterium sp. R1534 WT DNA were constructed into thepBluescriptII+(KS) or (SK) vector (Stratagene, La Jolla, USA) andtransformed into E. coli XL-1 blue (Stratagene) or JM109.

Media and growth conditions: Transformed E. coli were grown in Luriabroth (LB) at 37° C. with 100 mg Ampicillin (Amp)/ml for selection.Flavobacterium sp. R1534 WT was grown at 27° C. in medium containing 1%glucose, 1% tryptone (Difco Laboratories), 1% yeast extract (Difco),0.5% MgSO₄ 7H₂O and 3% NaCl.

Colony screening: Screening of the E. coli transformants was done by PCRbasically according to the method described by Zon et al. [Zon et al.,BioTechniques 7,696-698 (1989)] using the following primers:

(SEQ ID NO:38) Primer #7: 5′-CCTGGATGACGTGCTGGAATATTCC-3′ (SEQ ID NO:39)Primer #8: 5′-CAAGGCCCAGATCGCAGGCG-3′

Genomic DNA: A 50 ml overnight culture of Flavobacterium sp. R1534 wascentrifuged at 10,000 g for 10 minutes. The pellet was washed brieflywith 10 ml of lysis buffer (50 mM EDTA, 0.1M NaCl pH7.5), resuspended in4 ml of the same buffer sumplemented with 10 mg of lysozyme andincubated at 37° C. for 15 minutes. After addition of 0.3 ml ofN-Lauroyl sarcosine (20%) the incubation at 37° C. was continued foranother 15 minutes before the extraction of the DNA with phenol,phenol/chloroform and chloroform. The DNA was ethanol precipitated atroom temperature for 20 minutes in the presence of 0.3 M sodium acetate(pH 5.2), followed by centrifugation at 10,000 g for 15 minutes. Thepellet was rinsed with 70% ethanol, dried and resuspended in 1 ml of TE(10 mM Tris, 1 mM EDTA, pH 8.0).

All genomic DNA used in the southern blot analysis and cloningexperiments was dialysed against H₂O for 48 hours, using collodium bags(Sartorius, Germany), ethanol precipitated in the presence of 0.3 Msodium acetate and resuspended in H₂O.

Probe labelling: DNA probes were labeled with (a-³²P) dGTP (Amersham) byrandom-priming according to [Sambrook et al., s.a.].

Probes used to screen the mini-libraries: Probe 46F is a 119 bp fragmentobtained by PCR using primer #7 (SEQ ID NO: 38) and #8 (SEQ ID NO: 39)and Flavobacterium sp. R1534 genomic DNA as template. This probe wasproposed to be a fragment of the Flavobacterium sp. R1534 phytoenesynthase (crtB) gene, since it shows significant homology to thephytoene synthase genes from other species (e.g. E. uredovora, E.herbicola). Probe A is a BstXI-PstI fragment of 184 bp originating fromthe right arm of the insert of clone 85. Probe B is a 397 bp XhoI-NotIfragment obtained from the left end of the insert of clone 85. Probe Cis a 536 bp BgIII-PstI fragment from the right end of the insert ofclone 85. Probe D is a 376 bp KpnI-BstYI fragment isolated from theinsert of clone 59. The localization of the individual probes is shownin FIG. 6.

Oligonucleotide synthesis: The oligonucleotides used for PCR reactionsor for sequencing were synthesized with an Applied Biosystems 392 DNAsynthesizer.

Southern blot analysis: For hybridization experiments Flavobacterium sp.R1534 genomic DNA (3 mg) was digested with the appropiate restrictionenzymes and electrophoresed on a 0.75% agarose gel. The transfer toZeta-Probe blotting membranes (BIO-RAD), was done as described[Sourthern, E. M., J. Mol. Biol. 98, 503 (1975)]. Prehybridization andhybridization was in 7% SDS, 1% BSA (fraction V; Boehringer), 0.5MNa₂HPO₄, pH 7.2 at 65° C. After hybridization the membranes were washedtwice for 5 minutes in 2×SSC, 1% SDS at room temperature and twice for15 minutes in 0.1% SSC, 0.1% SDS at 65° C.

DNA sequence analysis: The sequence was determined by the dideoxy chaintermination technique [Sanger et al., Proc. Natl. Acad. Sci. USA 74,5463-5467 (1977)] using the Sequenase Kit (United States Biochemical).Both strands were completely sequenced and the sequence analyzed usingthe GCG sequence analysis software package (Version 8.0) by GeneticsComputer, Inc. [Devereux et al., Nucleic Acids. Res. 12, 387-395(1984)].

Analysis of carotenoids: E. coli XL-1 or JM109 cells (200-400 ml)carrying different plasmid constructs were grown for the times indicatedin the text, usually 24 to 60 hours, in LB suplemented with 100 mgAmpicillin/ml, in shake flasks at 37° C. and 220 rpm.

The carotenoids present in the microorganisms were extracted with anadequate volume of acetone using a rotation homogenizer (Polytron,Kinematica AG, CH-Luzern). The homogenate was the filtered through thesintered glass of a suction filter into a round bottom flask. Thefiltrate was evaporated by means of a rotation evaporator at 50° C.using a water-jet vacuum. For the zeaxanthin detection the residue wasdissolved in n-hexane/acetone (86:14) before analysis with a normalphaseHPLC as described in [Weber, S. in Analytical Methods for Vitamins andCarotenoids in Feed, Keller, H. E., Editor, 83-85 (1988)]. For thedetection of β-carotene and lycopene the evaporated extract wasdissolved in n-hexane/acetone (99:1) and analysed by HPLC as describedin [Hengartner et al., Helv. Chim. Acta 75, 1848-1865 (1992)].

EXAMPLE 2

Cloning of the Flavobacterium sp. R1534 Carotenoid Biosynthetic Genes

To identify and isolate DNA fragments carrying the genes of thecarotenoid biosynthesis pathway, we used the DNA fragment 46F (seemethods) to probe a Southern Blot carrying chromosomal DNA ofFlavobacterium sp. R1534 digested with different restriction enzymesFIG. 2. The 2.4 kb XhoI/PstI fragment hybridizing to the probe seemedthe most appropiate one to start with. Genomic Flavobacterium sp. R1534DNA was digested with XhoI/PstI and run on a 1% agarose gel. Accordingto a comigrating DNA marker, the region of about 2.4 kb was cut out ofthe gel and the DNA isolated. A XhoI/PstI mini library of Flavobacteriumsp. R1534 genomic DNA was constructed into XhoI-PstI sites ofpBluescriptIISK(+). One hundred E. coli XL1 transformants weresubsequently screened by PCR with primer #7 (SEQ ID NO: 38) and primer #8 (SEQ ID NO: 39), the same primers previously used to obtain the 119 bpfragment (46F). One positive transformant, named clone 85, was found.Sequencing of the insert revealed sequences not only homologous to thephytoene synthase (crtB) but also to the phytoene desaturase (crtI) ofboth Erwinia species herbicola and uredovora. Left and right handgenomic sequences of clone 85 were obtained by the same approach usingprobe A and probe B. Flavobacterium sp. R1534 genomic DNA was doubledigested with ClaI and Hind III and subjected to Southern analysis withprobe A and probe B. With probe A a ClaI/HindIII fragment of aprox. 1.8kb was identified (FIG. 3A), isolated and subcloned into theClaI/HindIII sites of pBluescriptIIKS (+). Screening of the E. coli XL1transformants with probe A gave 6 positive clones. The insert of one ofthese positives, clone 43-3, was sequenced and showed homology to theN-terminus of crtI genes and to the C-terminus of crtY genes of bothErwinia species mentioned above. With probe B an approx. 9.2 kbClaI/HindlIl fragment was detected (FIG. 3B), isolated and subclonedinto pBluescriptIIKS (+).

A screening of the transformants gave one positive, clone 51. Sequencingof the 5′ and 3′ of the insert, revealed that only the region close tothe HindIII site showed relevant homology to genes of the carotenoidbiosynthesis of the Erwinia species mentioned above (e.g. crtB gene andcrtE gene). The sequence around the ClaI site showed no homology toknown genes of the carotenoid biosynthesis pathway. Based on thisinformation and to facilitate further sequencing and construction work,the 4.2 kb BamHI/HindIII fragment of clone 51 was subcloned into therespective sites of pBluescriptIIKS(+) resulting in clone 2. Sequencingof the insert of this clone confirmed the presence of genes homologousto Erwinia sp crtB and crtE genes. These genes were located within 1.8kb from the HindIII site. The remaining 2.4 kb of this insert had nohomology to known carotenoid biosynthesis genes.

Additional genomic sequences downstream of the ClaI site were detectedusing probe C to hybridize to Flavobacterium sp. R1534 genomic DNAdigested with different restriction enzymes (see FIG. 4).

A SalI/HindIII fragment of 2.8 kb identified by Southern analysis wasisolated and subcloned into the HindIII/XhoI sites of pBluescriptIIKS(+). Screening of the E. coli XL1 transformants with probe A gave onepositive clone named clone 59. The insert of this clone confirmed thesequence of clone 43-3 and contained in addition sequences homologous tothe N-terminus of the crtY gene from other known lycopene cyclases. Toobtain the putative missing crtZ gene a Sau3AI partial digestion libraryof Flavobacterium sp. R1534 was constructed into the BamHI site ofpBluescriptIIKS(+). Screening of this library with probe D gave severalpositive clones. One transformant designated, clone 6a, had an insert of4.9 kb. Sequencing of the insert revealed besides the already knownsequences coding for crtB, crtI and crtY also the missing crtZ gene.Clone 7 g was isolated from a mini library carrying BclI/SphI fragmentsof R1534 (FIG. 5) and screened with probe D. The insert size of clone 7g is approx. 3 kb.

The six independent inserts of the clones described above coveringapprox. 14 kb of the Flavobacterium sp. R1534 genome are compiled inFIG. 6.

The determined sequence spanning from the BamHI site (position 1) tobase pair 8625 is shown FIG. 7.

Putative Protein Coding Regions of the Cloned R1534 Sequence

Computer analysis using the Codon Preference program of the GCG package,which recognizes protein coding regions by virtue of the similarity oftheir codon usage to a given codon frequency table, revealed eight openreading frames (ORFs) encoding putative proteins: a partial ORF from 1to 1165 (ORF-5) (SEQ ID NO: 41) coding for a polypeptide larger than41382 Da; an ORF coding for a polypeptide with a molecular weight of40081 Da from 1180 to 2352 (ORF-1) (SEQ ID NO: 40); an ORF coding for apolypeptide with a molecular weight of 31331 Da from 2521 to 3405(crtE); an ORF coding for a polypeptide with a molecular weight of 32615Da from 4316 to 3408 (crtB); an ORF coding for a polypeptide with amolecular weight of 54411 Da from 5797 to 4316 (crtI); an ORF coding fora polypeptide with a molecular weight of 42368 Da from 6942 to 5797(crtY); an ORF coding for a polypeptide with a molecular weight of 19282Da from 7448 to 6942 (crtZ); and an ORF coding for a polypeptide with amolecular weight of 19368 Da from 8315 to 7770 (ORF-16) (SEQ ID NO: 42);ORF-1 and crtE have the opposite transcriptional orientation from theothers (FIG. 6). The translation start sites of the ORFs crtI, crtY andcrtZ could clearly be determined based on the appropiately locatedsequences homologous to the Shine/Delgano (S/D) [Shine and Dalgarno,Proc. Natl. Acad. Sci. USA 71, 1342-1346 (1974)] consensus sequenceAGG-6-9N-ATG (FIG. 10) and the homology to the N-terminal sequences ofthe respective enzymes of E. herbicola and E. uredovora. The translationof the ORF crtB could potentially start from three closely spaced codonsATG (4316), ATG (4241) and ATG (4211). The first one, although nothaving the best S/D sequence of the three, gives a translation productwith the highest homology to the N-terminus of the E. herbicola and E.uredovora crtB protein, and is therefore the most likely translationstart site. The translation of ORF crtE could potentially start fromfive different start codons found within 150 bp: ATG (2389), ATG (2446),ATG (2473), ATG (2497) and ATG (2521). We believe that based on thefollowing observations, the ATG (2521) is the most likely transcriptionstart site of crtE: this ATG start codon is preceeded by the bestconsensus S/D sequence of all five putative start sites mentioned; andthe putative N-terminal amino acid sequence of the protein encoded hasthe highest homology to the N-terminus of the crtE enzymes of E.herbicola and E. uredovora;

Characteristics of the Crt Translational Initiation Sites and GeneProducts

The translational start sites of the five carotenoid biosynthesis genesare shown below and the possible ribosome binding sites are underlined.The genes crtZ, crtY, crtI and crtB are grouped so tightly that the TGAstop codon of the anterior gene overlaps the ATG of the following gene.Only three of the five genes (crtI, crtY and crtZ) fit with theconsensus for optimal S/D sequences. The boxed TGA sequence shows thestop condon of the anterior gene.

crtE (SEQ ID NO: 43)

crtB (SEQ ID NO: 44)

crtY (SEQ ID NO: 45)

crtI (SEQ ID NO: 46)

crtZ (SEQ ID NO: 47)

Amino Acid Sequences of Individual Crt Genes of Flavobacterium sp. R1534

All five ORFs of Flavobacterium sp. R1534 having homology to knowncarotenoid biosynthesis genes of other species are clustered in approx.5.2 kb of the sequence (FIG. 7) (SEQ ID NO: 1).

FFDP Synthase (crtE)

The amino acid (aa) sequence of the geranylgeranyl pyrophosphatesynthase (crtE gene product) consists of 295 aa and is shown in FIG. 8(SEQ ID NO: 2). This enzyme condenses farnesyl pyrophosphate andisopentenyl pyrophosphate in a 1′-4.

Phytoene Synthase (crtB)

This enzyme catalyzes two enzymatic steps. First it condenses in a headto head reaction two geranylgeranyl pyrophosphates (C20) to the C40carotenoid prephytoene. Second it rearanges the cyclopropylring ofprephytoene to phytoene. The 303 aa encoded by the crtB gene ofFlavobacterium sp. R1534 is shown in FIG. 9 (SEQ ID NO: 3).

Phytoene Desaturase (crtI)

The phytoene desaturase of Flavobacterium sp. R1534 consisting of 494aa, shown in FIG. 10 (SEQ ID NO: 4), performs like the crtI enzyme of E.herbicola and E. uredovora, four desaturation steps, converting thenon-coloured carotenoid phytoene to the red coloured lycopene.

Lycopene Cyclase (crtY)

The crtY gene product of Flavobacterium sp. R1534 is sufficient tointroduce the b-ionone rings at both sides of lycopene to obtainβ-carotene. The lycopene cyclase of Flavobacterium sp. R1534 consists of382 aa (FIG. 11) (SEQ ID NO: 5).

β-carotene Hydroxylase (crtZ)

The gene product of crtZ consisting of 169 aa (FIG. 12) (SEQ ID NO: 6)and hydroxylates β-carotene to the xanthophyll zeaxanthin.

Putative Enzymatic Functions of the ORF's (Orf-1 (SEQ ID NO: 40), Orf-5(SEQ ID NO: 41) and Orf-16 (SEQ ID NO: 42))

The orf-1 (SEQ ID NO: 40) has at the aa level over 40% identity toacetoacetyl-CoA thiolases of different organisms (e.g. Candidatropicalis, human, rat). This gene is therefore most likely a putativeacetoacetyl-CoA thiolase (acetyl-CoA acetyltransferase), which condensestwo molecules of acetyl-CoA to Acetoacetyl-CoA. Condensation ofacetoacetyl-CoA with a third acetyl-CoA by the HMG-CoA synthase formsβ-hydroxy-β-methylglutaryl-CoA (HMG-CoA). This compound is part of themevalonate pathway which produces besides sterols also numerous kinds ofisoprenoids with diverse cellular functions. In bacteria and plants, theisoprenoid pathway is also able to synthesize some unique products likecarotenoids, growth regulators (e.g. in plants gibberellins and abcissicacid) and sencodary metabolites like phytoalexins [Riou et al., Gene148, 293-297 (1994)].

The orf-5 (SEQ ID NO: 41) has a low homology of approx. 30%, to theamino acid sequence of polyketide synthases from different streptomyces(e.g. S. violaceoruber, S. cinnamonensis). These antibiotic synthesizingenzymes (polyketide synthases), have been classified into two groups.Type-I polyketide synthases are large multifunctional proteins, whereastype-II polyketide synthases are multiprotein complexes composed ofseveral individual proteins involved in the subreactions of thepolyketide synthesis [Bibb, et al. Gene 142, 31-39 (1994)].

The putative protein encoded by the orf-16 (SEQ ID NO: 42) has at the aalevel an identity of 42% when compared to the soluble hydrogenasesubunit of Anabaena cylindrica.

Functional Assignment of the ORF 's (crtE, crtB, crtI, crtY and crtZ) toEnzymatic Activities of the Carotenoid Biosynthesis Pathway

The biochemical assignment of the gene products of the different ORF'swere revealed by analyzing carotenoid accumulation in E. coli hoststrains that were transformed with deleted variants of theFlavobacterium sp. gene cluster and thus expressed not all of the crtgenes (FIG. 13).

Three different plasmid were constructed: pLyco, p59-2 and pZea4.Plasmid p59-2 was obtained by subcloning the HindIII/BamHI fragment ofclone 2 into the HindIII/BamHI sites of clone 59. p59-2 carries theORF's of the crtE, crtB, crtI and crtY gene and should lead to theproduction of β-carotene. pLyco was obtained by deleting the KpnI/KpnIfragment, coding for approx. one half (N-terminus) of the crtY gene,from the p59-2 plasmid. E. coli cells transformed with pLyco, andtherefore having a truncated non-functional crtY gene, should producelycopene, the precursor of β-carotene. pZea4 was constructed by ligationof the AscI-SpeI fragment of p59-2, containing the crtE, crtB, crtI andmost of the crtY gene with the AscI/XbaI fragment of clone 6a,containing the sequences to complete the crtY gene and the crtZ gene.pZea4 [for complete sequence see FIG. 24 (SEQ ID NO: 27); nucleotides 1to 683 result from pBluescriptIIKS(+), nucleotides 684 to 8961 fromFlavobacterium R1534 WT genome, nucleotides 8962 to 11233 frompBluescriptIIKS(+)] has therefore all five ORF's of the zeaxanthinbiosynthesis pathway. Plasmid pZea4 has been deposited on May 25, 1995at the DSM-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH(Germany) under accession No. DSM 10012. E. coli cells transformed withthis latter plasmid should therefore produce zeaxanthin. For thedetection of the carotenoid produced, transformants were grown for 48hours in shake flasks and then subjected to carotenoid analysis asdescribed in the methods section. FIG. 13 summarizes the differentinserts of the plasmids described above, and the main carotenoiddetected in the cells.

As expected the pLyco carrying E. coli cells produced lycopene, thosecarrying p59-2 produced β-carotene (all-E,9-Z,13-Z) and the cells havingthe pZea4 construct produced zeaxanthin. This confirms that all thenecessary genes of Flavobacterium sp. R1534 for the synthesis ofzeaxanthin or their precursors (phytoene, lycopene and β-carotene) werecloned.

EXAMPLE 3

Materials and Methods Used for Expression of Carotenoid SynthesizingEnzymes

Bacterial strains and plasmids: The vectors pBluescriptIIKS (+) or (−)(Stratagene, La Jolla, USA) and pUC18 [Vieira and Messing, Gene 19,259-268 (1982); Norrander et al., Gene 26, 101-106 (1983)] were used forcloning in different E. coli strains, like XL-1 blue (Stratagene), TG1or JM109. In all B. subtilis transformations, strain 1012 was used.Plasmids pHP13 [Haima et al., Mol. Gen. Genet. 209, 335-342 (1987)] andp602/22 [LeGrice, S. F. J. in Gene Expression Technology, Goeddel, D.V., Editor, 201-214 (1990)] are Gram (+)/(−) shuttle vectors able toreplicate in B. subtilis and E. coli cells. Plasmid p205 contains thevegI promoter cloned into the SmaI site of pUC18. Plasmid pXI12 is anintegration vector for the constitutive expression of genes in B.subtilis [Haiker et al., in 7th Int. Symposium on the Genetics ofIndustrial Microorganisms, Jun. 26-Jul. 1, 1994. Montreal, Quebec,Canada (1994)]. Plasmid pBEST501 [ltaya et al., Nucleic Acids Res. 17(11), 4410 (1989)] contains the neomycin resistance gene cassetteoriginating from the plasmid pUB110 (GenBank entry: M19465) of S. aureus[McKenzie et al., Plasmid 15, 93-103 (1986); McKenzie et al., Plasmid17, 83-84 (1987)]. This neomycin gene has been shown to work as aselection marker when present in a single copy in the genome of B.subtilis, Plasmid pC194 (ATCC 37034)(GenBank entry: L08860) originatesfrom S. aureuis [Horinouchi and Weisblaum, J. Bacteriol. 150, 815-825(1982)] and contains the chloramphenicol acetyltransferase gene.

Media and growth conditions: E. coli were grown in Luria broth (LB) at37° C. with 100 mg Ampicillin (Amp)/ml for selection. B. subtilis cellswere grown in VY-medium supplemented with either erythromycin (1 mg/ml),neomycin (5-180 mg/ml) or chloramphenicol (10-80 mg/ml).

Transformation: E. coli transformations were done by electroporationusing the Gene-pulser device of BIO-RAD (Hercules, Calif., USA) with thefollowing parameters (200 W, 250 mFD, 2.5V). B. subtilis transformationswere done basically according to the standard procedure method 2.8described by [Cutting and Vander Horn in Molecular Biological Methodsfor Bacillus, Harwood, C. R. and Cutting, S. M., Editor, John Wiley &Sons: Chichester, England. 61-74 (1990)].

Colony screening: Bacterial colony screening was done as described by[Zon et al., s.a.].

Oligonucleotide synthesis: The oligonucleotides used for PCR reactionsor for sequencing were synthesized with an Applied Biosystems 392 DNAsynthesizer.

PCR reactions: The PCR reactions were performed using either the UlTmaDNA polymerase (Perkin Elmer Cetus) or the Pfu Vent polymerase (NewEngland Biolabs) according to the manufacturers instructions. A typical50 ml PCR reaction contained: 100 ng of template DNA, 10 pM of each ofthe primers, all four dNTP's (final conc. 300 mM), MgCl₂ ₍when UlTmapolymerase was used; final conc. 2 mM), 1×UlTma reaction buffer or 1×Pfubuffer (supplied by the manufacturer). All components of the reactionwith the exception of the DNA polymerase were incubated at 95° C. for 2min. followed by the cycles indicated in the respective section (seebelow). In all reactions a hot start was made, by adding the polymerasein the first round of the cycle during the 72° C. elongation step. Atthe end of the PCR reaction an aliquot was analysed on 1% agarose gel,before extracting once with phenol/chloroform. The amplified fragment inthe aqueous phase was precipitated with 1/10 of a 3M NaAcetate solutionand two volumes of Ethanol. After centrifugation for 5 min. at 12000rpm, the pellet was resuspended in an adequate volume of H₂O, typically40 ml, before digestion with the indicated restriction enzymes wasperformed. After the digestion the mixture was separated on a 1% lowmelting point agarose. The PCR product of the expected size were excisedfrom the agarose and purified using the glass beads method (GENECLEANKIT, Bio 101, Vista Calif., USA) when the fragments were above 400 bp ordirectly spun out of the gel when the fragments were shorter than 400bp, as described by [Heery et al., TIBS 6 (6), 173 (1990)].

Oligos Used for Gene Amplification and Site Directed Mutagenesis

All PCR reactions performed to allow the construction of the differentplasmids are described below. All the primers used are summarized inFIG. 14.

Primers #100 (SEQ ID NO: 7) and #101 (SEQ ID NO: 8) were used in a PCRreaction to amplify the complete crtE gene having a SpeI restrictionsite and an artificial ribosomal binding site (RBS) upstream of thetranscription start site of this gene. At the 3′ end of the amplifiedfragment, two unique restriction sites were introduced, an AvrII and aSmaI site, to facilitate the further cloning steps. The PCR reaction wasdone with UlTma polymerase using the following conditions for theamplification: 5 cycles with the profile: 95° C., 1 min./60° C., 45sec./72° C., 1 min. and 20 cycles with the profile: 95° C., 1 min./72°C., 1 min. Plasmid pBIIKS(+)-clone2 served as template DNA. The finalPCR product was digested with SpeI and SmaI and isolated using theGENECLEAN KIT. The size of the fragment was approx. 910 bp.

Primers #104 (SEQ ID NO: 9) and #105 (SEQ ID NO: 10) were used in a PCRreaction to amplify the crtZ gene from the translation start till theSalI restriction site, located in the coding sequence of this gene. Atthe 5′ end of the crtZ gene an EcoRI, a synthetic RBS and a NdeI sitewas introduced. The PCR conditions were as described above. PlasmidpBIIKS(+)-clone 6a served as template DNA and the final PCR product wasdigested with EcoRI and SalI. Isolation of the fragment of approx. 480bp was done with the GENECLEAN KIT.

Primers MUT1 (SEQ ID NO: 11) and MUT5 (SEQ ID NO: 14) were used toamplify the complete crtY gene. At the 5′ end, the last 23 nucleotidesof the crtZ gene including the SalI site are present, followed by anartificial RBS preceding the translation start site of the crtY gene.The artificial RBS created includes a PmlI restriction site. The 3′ endof the amplified fragment contains 22 nucleotides of the crtI gene,preceded by a newly created artificial RBS which contains a MunIrestriction site. The conditions used for the PCR reaction were asdescribed above using the following cycling profile: 5 rounds of 95° C.,45 sec./60° C., 45 sec./72° C., 75 sec. followed by 22 cycles with theprofile: 95° C., 45 sec./66° C., 45 sec./72° C., 75 sec. PlasmidpXI12-ZYIB-EINV4 served as template for the Pfu Vent polymerase. The PCRproduct of 1225 bp was made blunt and cloned into the SmaI site ofpUC18, using the Sure-Clone Kit (Pharmacia) according to themanufacturer.

Primers MUT2 (SEQ ID NO: 15) and MUT6 (SEQ ID NO: 15) were used toamplify the complete crtI gene. At the 5′ the last 23 nucleotides of thecrtY gene are present, followed by an artificial RBS which precedes thetranslation start site of the crtI gene. The new RBS created, includes aMunI restriction site. The 3′ end of the amplified fragment contains theartificial RBS upstream of the crtB gene including a BamHI restrictionsite. The conditions used for the PCR reaction were basically asdescribed above including the following cycling profile: 5 rounds of 95°C., 30 sec./60° C., 30 sec./72° C., 75 sec., followed by 25 cycles withthe profile: 95° C., 30 sec./66° C., 30 sec./72° C., 75 sec. PlasmidpXI12-ZYIB-EINV4 served as template for the Pfu Vent polymerase. For thefurther cloning steps the PCR product of 1541 bp was digested with MunIand BamHI.

Primers MUT3 (SEQ ID NO: 13) and CAR17 (SEQ ID NO: 16) were used toamplify the N-terminus of the crtB gene. At the 5′ the last 28nucleotides of the crtI gene are present followed by an artificial RBS,preceding the translation start site of the crtB gene. This new createdRBS, includes a BamHI restriction site. The amplified fragment, namedPCR-F contains also the HindIII restriction site located at theN-terminus of the crtB gene. The conditions used for the PCR reactionwere as described elsewhere in the text, including the following cyclingprofile: 5 rounds of 95° C., 30 sec./58° C., 30 sec./72° C., 20 sec.followed by 25 cycles with the profile; 95° C., 30 sec./60° C., 30sec./72° C., 20 sec. Plasmid pXI12-ZYIB-EINV4 for the Pfu Ventpolymerase. The PCR product of approx. 160 bp was digested with BamHIand HindIII.

Oligos Used to Amplify the Chloramphenicol Resistance Gene (Cat)

Primers CAT3 (SEQ ID NO: 17) and CAT4 (SEQ ID NO: 18) were used toamplify the chloramphenicol resistance gene of pC194 (ATCC 37034)[Horinouchi and Weisblum, s.a.] a R-plasmid found in S. aureus. Theconditions used for the PCR reaction were as described previouslyincluding the following cycling profile: 5 rounds of 95° C., 60 sec./50°C., 60 sec./72° C., 2 min. followed by 20 cycles with the profile: 95°C., 60 sec./60° C., 60 sec./72° C., 2 min. Plasmid pC198 served astemplate for the Pfu Vent polymerase. The PCR product of approx. 1050 bpwas digested with EcoRI and AatII.

Oligos used to generate linkers: Linkers were obtained by adding 90 ngof each of the two corresponding primers into an Eppendorf tube. Themixture was dried in a speed vac and the pellet resuspended in 1×Ligation buffer (Boehringer, Mannheim, Germany). The solution wasincubated at 50° C. for 3 min. before cooling down to RT, to allow theprimers to hybridize properly. The linker were now ready to be ligatedinto the appropriate sites. All the oligos used to generate linkers areshown in FIG. 15.

Primers CS1 (SEQ ID NO: 19) and CS2 (SEQ ID NO: 20) were used to form alinker containing the following restrictions sites HindIII, AflII, ScaI,XbaI, PmeI and EcoRI.

Primers MUT7 (SEQ ID NO: 21) and MUT8 (SEQ ID NO: 22) were used to forma linker containing the restriction sites SalI, AvrII, PmlI, MluI, MunI,BamHI, SphI and HindIII.

Primers MUT9 (SEQ ID NO: 23) and MUT10 (SEQ ID NO: 24) were used tointroduce an artificial RBS upstream of crtY.

Primers MUT11 (SEQ ID NO: 25) and MUT12 (SEQ ID NO: 26) were used tointroduce an artificial RBS upstream of crtE.

Isolation of RNA: Total RNA was prepared from log phase growing B.subtilis according to the method described by [Maes and Messens, NucleicAcids Res. 20 (16), 4374 (1992)].

Northern blot analysis: For hybridization experiments up to 30 mg of B.subtilis RNA was electrophoreses on a 1% agarose gel made up in 1×MOPSand 0.66 M formaldehyde. Transfer to Zeta-Probe blotting membranes(BIO-RAD), UV cross-linking, pre-hybridization and hybridization wasdone as described elsewhere in [Farrell, J. R. E., RNA Methodologies. Alaboratory Guide for isolation and characterization. San Diego, USA:Academic Press (1993)]. The washing conditions used were: 2×20 min. in2×SSPE/0.1% SDS followed by 1×20 min. in 0.1% SSPE/0.1% SDS at 65° C.Northern blots were then analyzed either by a Phosphorimager (MolecularDynamics) or by autoradiography on X-ray films from Kodak.

Isolation of genomic DNA: B. subtilis genomic DNA was isolated from 25ml overnight cultures according to the standard procedure method 2.6described by [13].

Southern blot analysis: For hybridization experiments B. subtilisgenomic DNA (3 mg) was digested with the appropriate restriction enzymesand electrophoresed on a 0.75% agarose gel. The transfer to Zeta-Probeblotting membranes (BIO-RAD), was done as described [Southern, E. M.,s.a.]. Prehybridization and hybridization was in 7%SDS, 1% BSA (fractionV; Boehringer), 0.5M Na₂HPO₄, pH 7.2 at 65° C. After hybridization themembranes were washed twice for 5 min. in 2×SSC, 1% SDS at roomtemperature and twice for 15 min. in 0.1% SSC, 0.1% SDS at 65° C.Southern blots were then analyzed either by a Phosphorimager (MolecularDynamics) or by autoradiography on X-ray films from Kodak.

DNA sequence analysis: The sequence was determined by the dideoxy chaintermination technique [Sanger et al., s.a.] using the Sequenase KitVersion 1.0 (United States Biochemical). Sequence analysis were doneusing the GCG sequence analysis software package (Version 8.0) byGenetics Computer, Inc. [Devereux et al., s.a.].

Gene amplification in B. subtilis: To amplify the copy number of theSFCO in B. subtilis transformants, a single colony was inoculated in 15ml VY-medium supplemented with 1.5% glucose and 0.02 mg chloramphenicolor neomycin/ml, dependend on the antibiotic resistance gene present inthe amplifiable structure (see results and discussion). The next day 750ml of this culture were used to inoculate 13 ml VY-medium containing1.5% glucose supplemented with (60, 80, 120 and 150 mg/ml) for the catresistant mutants, or 160 mg/ml and 180 mg/ml for the neomycin resistantmutants). The cultures were grown overnight and the next day 50 ml ofdifferent dilutions (1:20, 1:400, 1:8000, 1:160′000) were plated on VYagar plates with the appropriate antibiotic concentration. Large singlecolonies were then further analyzed to determine the number of copiesand the amount of carotenoids produced.

Analysis of carotenoids: E. coli or B. subtilis transformants (200-400ml) were grown for the times indicated in the text, usually 24 to 72hours, in LB-medium or VY-medium, respectively, supplemented withantibiotics, in shake flasks at 37° C. and 220 rpm.

The carotenoids produced by the microorganisms were extracted with anadequate volume of acetone using a rotation homogenizer (Polytron,Kinematica AG, CH-Luzern). The homogenate was the filtered through thesintered glass of a suction filter into a round bottom flask. Thefiltrate was evaporated by means of a rotation evaporator at 50° C.using a water-jet vacuum. For the zeaxanthin detection the residue wasdissolved in n-hexane/acetone (86:14) before analysis with a normalphaseHPLC as described in [Weber, S., s.a.]. For the detection of β-caroteneand lycopene the evaporated extract was dissolved in n-hexane/acetone(99:1) and analysed by HPLC as described in Hengartner et al., s.a.].

EXAMPLE 4

Carotenoid Production in E. coli

The biochemical assignment of the gene products of the different openreading frames (ORF's) of the carotenoid biosynthesis cluster ofFlavobacterium sp. were revealed by analyzing the carotenoidaccumulation in E. coli host strains, transformed with plasmids carryingdeletions of the Flavobacterium sp. gene cluster, and thus lacking someof the crt gene products. Similar functional assays in E. coli have beendescribed by other authors [Misawa et al., s.a.; Perry et al., J.Bacteriol., 168, 607-612 (1986); Hundle, et al., Molecular and GeneralGenetics 254 (4), 406-416 (1994)]. Three different plasmid pLyco,pBIIKS(+)-clone59-2 and pZea4 were constructed from the three genomicisolates pBIIKS(+)-clone2, pBIIKS(+)-clone59 and pBIIKS(+)-clone6a (seeFIG. 16).

Plasmid pBIIKS(+)-clone59-2 was obtained by subcloning the HindIII/BamHIfragment of pBIIKS(+)-clone 2 into the HindIII/BamHI sites ofpBIIKS(+)-clone59. The resulting plasmid pBIIKS(+)-clone59-2 carries thecomplete ORF's of the crtE, crtB, crtI and crtY gene and should lead tothe production of β-carotene. pLyco was obtained by deleting theKpnI/KpnI fragment, coding for approx. one half (N-terminus) of the crtYgene, from the plasmid pBIIKS(+)-clone59-2. E. coli cells transformedwith pLyco, and therefore having a truncated non-functional crtY gene,should produce lycopene, the precursor of β-carotene. pZea4 wasconstructed by ligation of the AscI-SpeI fragment ofpBIIKS(+)-clone59-2, containing the crtE, crtB, crtI and most of thecrtY gene with the AscI/XbaI fragment of clone 6a, containing the crtZgene and sequences to complete the truncated crtY gene mentioned above.pZea4 has therefore all five ORF's of the zeaxanthin biosynthesispathway. E. coli cells transformed with this latter plasmid shouldtherefore produce zeaxanthin. For the detection of the carotenoidproduced, transformants were grown for 43 hours in shake flasks and thensubjected to carotenoid analysis as described in the methods section.FIG. 16 summarizes the construction of the plasmids described above.

As expected the pLyco carrying E. coli cells produced lycopene, thosecarrying pBIIKS(+)-clone59-2 produced β-carotene (all-E,9-Z,13-Z) andthe cells having the pZea4 construct produced zeaxanthin. This confirmsthat we have cloned all the necessary genes of Flavobacterium sp. R1534for the synthesis of zeaxanthin or their precursors (phytoene, lycopeneand β-carotene). The production levels obtained are shown in table 1.

TABLE 1 Carotenoid content of E. coli transformants, carrying theplasmids pLyco, pBIIKS(+)-clone59-2 and pZea4, after 43 hours of culturein shake flasks. The values indicated show the carotenoid content in %of the total dry cell mass (200 ml). ND = not detectable. plasmid hostzeaxanthin β-χαρoτενε lycopene pLyco E. coli JM109 ND ND 0.05%pBIIKS(+)- ″ ND 0.03% ND clone59-2 pZea4 ″ 0.033% 0.0009% ND

EXAMPLES 5

Carotenoid Production in B. subtilis

In a first approach to produce carotenoids in B. subtilis, we cloned thecarotenoid biosynthesis genes of Flavobacterium into the Gram (+)/(−)shuttle vectors p602/22, a derivative of p602/20 [LeGrice, S. F. J.,s.a.]. The assembling of the final construct p602-CARVEG-E, begins witha triple ligation of fragments PvuII-AvrII of pZea4(del654-3028) and theAvrII-EcoRI fragment from plasmid pBIIKS(+)-clone6a, into the EcoRI andScaI sites of the vector p602/22. The plasmid pZea4(del654-3028) hadbeen obtained by digesting pZea4 with SacI and EspI. The protruding andrecessed ends were made blunt with Klenow enzyme and religated.Construct pZea4(del654-3028) lacks most of the sequence upstream of crtEgene, which are not needed for the carotenoid biosynthesis. The plasmidp602-CAR has approx. 6.7 kb of genomic Flavobacterium R1534 DNAcontaining besides all five carotenoid genes (approx. 4.9 kb),additional genomic DNA of 1.2 kb, located upstream of the crtZtranslation start site and further 200 bp, located upstream of crtEtranscription start. The crtZ, crtY, crtI and crtB genes were cloneddownstream of the P_(N25/0) promoter, a regulatable E. colibacteriophage T5 promoter derivative, fused to a lac operator element,which is functional in B. subtilis [LeGrice, S. F. J., s.a.]. It isobvious that in the p602CAR construct, the distance of over 1200 bpbetween the P_(N25/0) promoter and the transcription start site of crtZis not optimal and will be improved at a later stage. An outline of thep602CAR construction is shown in FIG. 17. To ensure transcription of thecrtE gene in B. subtilis, the vegI promoter [Moran et al., Mol. Gen.Genet. 186, 339-346 (1982); LeGrice et al., Mol. Gen. Genet. 204,229-236 (1986)] was introduced upstream of this gene, resulting in theplasmid construct p602-CARVEG-E. The vegI promoter, which originatesfrom siteI of the veg promoter complex described by [LeGrice et al.,s.a.] has been shown to be functional in E. coli [Moran et al., s.a.].To obtain this new construct, the plasmid p602CAR was digested with SalIand HindIII, and the fragment containing the complete crtE gene and mostof the crtB coding sequence, was subcloned into the XhoI and HindIIIsites of plasmid p205. The resulting plasmid p205CAR contains the crtEgene just downstream of the PvegI promoter. To reconstitute thecarotenoid gene cluster of Flavobacterium sp. The following three pieceswere isolated: PmeI/HindIII fragment of p205CAR, the HindIII/XbaIfragment and the EcoRI/HindIII fragment of p602CAR and ligated into theEcoRI and XbaI sites of pBluescriptIIKS(+), resulting in the constructpBIIKS(+)-CARVEG-E. Isolation of the EcoRI-XbaI fragment of this latterplasmid and ligation into the EcoRI and XbaI sites of p602/22 gives aplasmid similar to p602CAR but having the crtE gene driven by the PvegIpromoter. All the construction steps to get the plasmid p602CARVEG-E areoutlined in FIG. 18. E. coli TG1 cells transformed with this plasmidsynthesized zeaxanthin. In contrast B. subtilis strain 1012 transformedwith the same constructs did not produce any carotenoids. Analysis ofseveral zeaxanthin negative B. subtilis transformants always revealed,that the transformed plasmids had undergone severe deletions. Thisinstability could be due to the large size of the constructs.

In order to obtain a stable construct in B. subtilis, the carotenoidgenes were cloned into the Gram (+)/(−) shuttle vector pHP13 constructedby [Haima et al., s.a.]. The stability problems were thought to beomitted by 1) reducing the size of the cloned insert which carries thecarotenoid genes and 2) reversing the orientation of the crtE gene andthus only requiring one promoter for the expression of all five genes,instead of two, like in the previous constructs. Furthermore, theability of cells transformed by such a plasmid carrying the syntheticFlavobacterium carotenoid operon (SFCO), to produce carotenoids, wouldanswer the question if a modular approach is feasible. FIG. 19summarizes all the construction steps and intermediate plasmids made toget the final construct pHP13-2PNZYIB-EINV. Briefly: To facilitate thefollowing constructions, a vector pHP13-2 was made, by introducing asynthetic linker obtained with primer CS1 (SEQ ID NO: 19) and CS2 (SEQID NO: 20), between the HindIII and EcoRI sites of the shuttle vectorpHP13. The intermediate construct pHP13-2CARVEG-E was constructed bysubcloning the AflII-XbaI fragment of p602CARVEG-E into the AflII andXbaI sites of pHP13-2. The next step consisted in the inversion of crtEgene, by removing XbaI and AvrII fragment containing the original crtEgene and replacing it with the XbaI-AvrII fragment of plasmidpBIIKS(+)-PCRRBScrtE. The resulting plasmid was namedpHP13-2CARZYIB-EINV and represented the first construction with afunctional SFCO. The intermediate construct pBIIKS(+)-PCRRBScrtEmentioned above, was obtained by digesting the PCR product generatedwith primers #100 (SEQ ID NO: 7) and #101 (SEQ ID NO: 8) with SpeI andSmaI and ligating into the SpeI and SmaI sites of pBluescriptIIKS(+). Inorder to get the crtZ transcription start close to the promoterP_(N25/0) a triple ligation was done with the BamHI-SalI fragment ofpHP13-2CARZYIB-EINV (contains four of the five carotenoid genes), theBamHI-EcoRI fragment of the same plasmid containing the P_(N25/0)promoter and the EcoRI-SalI fragment of pBIIKS(+)-PCRRBScrtZ, havingmost of the crtZ gene preceded by a synthetic RBS. The aforementionedplasmid pBIISK(+)-PCRRBScrtZ was obtained by digesting the PCR productamplified with primers #104 (SEQ ID NO: 9) and #105 (SEQ ID NO: 10) withEcoRI and SalI and ligating into the EcoRI and SalI sites ofpBluescriptIISK(+). In the resulting vector pHP13-2PN25ZYIB-EINV, theSFCO is driven by the bacteriophage T5 promoter P_(N25/0), which shouldbe constitutively expressed, due to the absence of a functional lacrepressor in the construct [Peschke and Beuk, J. Mol. Biol. 186, 547-555(1985)]. E. coli TG1 cells transformed with this construct producedzeaxanthin. Nevertheless, when this plasmid was transformed into B.subtilis, no carotenoid production could be detected. Analysis of theplasmids of these transformants showed severe deletions, pointingtowards instability problems, similar to the observations made with theaforementioned plasmids.

EXAMPLES 6

Chromosome Integration Constructs

Due to the instability observed with the previous constructs we decidedto integrate the carotenoid biosynthesis genes of Flavobacterium sp.into the genome of B. subtilis using the integration/expression vectorpXI12. This vector allows the constitutive expression of whole operonsafter integration into the levan-sucrase gene (sacB) of the B. subtilisgenome. The constitutive expression is driven by the vegI promoter andresults in medium level expression. The plasmid pXI12-ZYIB-EINV4containing the synthetic Flavobacterium carotenoid operon (SFCO) wasconstructed as follows: the NdeI-HincII fragment of pBIISK(+)-PCRRBScrtZwas cloned into the NdeI and SmaI sites of pXI12 and the resultingplasmid was named pXI12-PCRcrtZ. In the next step, the BstEII-PmeIfragment of pHP13-2PN25ZYIB-EINV was ligated to the BstEII-PmeI fragmentof pXI12-PCRcrtZ (see FIG. 20). B. subtilis transformed with theresulting construct pXI12-ZYIB-EINV4 can integrate the CAR genes eithervia a Campbell type reaction or via a reciprocal recombination. Onetransformant, BS1012::ZYIB-EINV4, having a reciprocal recombination ofthe carotenoid biosynthesis genes into the levan-sucrase gene wasfurther analyzed (FIG. 21). Although this strain did not synthesizecarotenoids, RNA analysis by Northern blots showed the presence ofspecific polycistronic mRNA's of 5.4 kb and 4.2 kb when hybridized toprobe A (see FIG. 21, panel B). Whereas the larger mRNA has the expectedmessage size, the origin of the shorter mRNA was unclear. Hybridizationof the same Northern blot to probe B only detected the large mRNAfragment, pointing towards a premature termination of the transcriptionat the end of the crtB gene. The presence of a termination signal atthis location would make sense, since in the original operonorganisation in the Flavobacterium sp. R1534 genome, the crtE and thecrtB genes are facing each other. With this constellation atranscription termination signal at the 5′ end of crtB would make sense,in order to avoid the synthesis of anti-sense RNA which could interferewith the mRNA transcript of the crtE gene. Since this region has beenchanged considerably with respect to the wild type situation, thesequences constituting this terminator may also have been alteredresulting in a “leaky” terminator. Western blot analysis using antiseraagainst the different crt enzymes of the carotenoid pathway, pointedtowards the possibility that the ribosomal binding sites might beresponsible for the lack of carotenoid synthesis. Out of the five genesintroduced only the product of crtZ, the β-carotene hydroxylase wasdetectable. This is the only gene preceded by a RBS site, originatingfrom the pXI12 vector, known to be functional in B. subtilis. Basepairing interactions between a mRNA's Shine-Dalgarno sequence [Shine andDelagarno, s. a.] and the 16S rRNA, which permits the ribosome to selectthe proper initiation site, have been proposed by [McLaughlin et al., J.Biol. Chem. 256,11283-11291 (1981)] to be much more stable inGram-positive organisms (B. subtilis) than in Gram-negative organisms(E. coli). In order to obtain highly stable complexes we exchanged theRBS sites of the Gram-negative Flavobacterium sp., preceding each of thegenes crtY, crtI, crtB and crtE, with synthetic RBS's which weredesigned complementary to the 3′ end of the B. subtilis 16S rRNA (seetable 2). This exchange should allow an effective translation initiationof the different carotenoid genes in B. subtilis. The strategy chosen toconstruct this pXI12-ZYIB-EINV4MUTRBS2C, containing all four alteredsites is summarized in FIG. 20. In order to facilitate the furthercloning steps in pBluescriptIIKS(+), additional restriction sites wereintroduced using the linker obtained with primer MUT7 and MUT8, clonedbetween the SalI and HindIII sites of said vector. The new resultingconstruct pBIIKS(+)-LINKER78 had the following restriction sitesintroduced: AvrII, PmlI, MulI, MunI, BamHI and SphI. The generalapproach chosen to create the synthetic RBS's upstream of the differentcarotenoid genes, was done using a combination of PCR based mutagenesis,where the genes were reconstructed using defined primers carrying themodified RBS sites, or using synthetic linkers having such sequences.Reconstitution of the RBS preceding the crtI and crtB genes was done byamplifying the crtI gene with the primers MUT2 (SEQ ID NO: 12) and MUT6(SEQ ID NO: 15), which include the appropriate altered RBS sites. ThePCR-I fragment obtained was digested with MunI and BamHI and ligatedinto the MunI and BamHI sites of pBIIKS(+)-LINKER78. The resultingintermediate construct was named pBIIKS(+)-LINKER78PCRI. Reconstitutionof the RBS preceding the crtB gene was done using a small PCR fragmentobtained with primer MUT3 (SEQ ID NO: 13), carrying the altered RBS siteupstream of crtB, and primer CAR17 (SEQ ID NO: 16). The amplified PCR-Ffragment was digested with BamHI and HindIII and sub cloned into theBamHI and HindIII sites of pBIIKS(+)-LINKER78, resulting in theconstruct pBIIKS(+)-LINKER78PCRF. The PCR-I fragment was cut out ofpBIIKS(+)-LINKER78PCRI with BamHI and SapI and ligated into the BamHIand SapI sites of pBIIKS(+)-LINKER78PCRF. The resulting plasmidpBIIKS(+)-LINKER78PCRFI has the PCR-I fragment fused to the PCR-Ffragment. This construct was cut with SalI and PmlI and a syntheticlinker obtained by annealing of primer MUT9 (SEQ ID NO: 23) and MUT10(SEQ ID NO: 24) was introduced. This latter step was done to facilitatethe upcoming replacement of the original Flavobacterium RBS in the abovementioned construct. The resulting plasmid was namedpBIIKS(+)-LINKER78PCRFIA. Assembling of the synthetic RBS's precedingthe crtY and crtI genes was done by PCR, using primers MUT1 (SEQ ID NO:11) and MUT5 (SEQ ID NO: 14). The amplified fragment PCR-G was madeblunt end before cloning into the SmaI site of pUC18, resulting inconstruct pUC18-PCR-G. The next step was the cloning of the PCR-Gfragment between the PCR-A and PCR-I fragments. For this purpose thePCR-G was isolated from pUC18-PCR-G by digesting with MunI and PmlI andligated into the MunI and PmlI sites of pBIIKS(+)-LINKER78PCRFIA. Thisconstruct contains all four fragments, PCR-F, PCR-I, PCR-G and PCR-A,assembled adjacent to each other and containing three of the fourartificial RBS sites (crtY, crtI and crtB). The exchange of theFlavobacterium RBS's preceding the genes crtY, crtI and crtB bysynthetic ones, was done by replacing the HindIII-SalI fragment ofplasmid pXI12-ZYIB-EINV4 with the HindIII-SalI fragment of plasmidpBIIKS(+)-LINKER78PCRFIGA. The resulting plasmid pXI12-ZYIB-EINV4MUTRBSC was subsequently transformed into E. coli TG1 cells and B.subtilis 1012. The production of zeaxanthin by these cells confirmedthat the PCR amplified genes where functional. The B. subtilis strainobtained was named BS1012::SFCO1. The last Flavobacterium RBS to beexchanged was the one preceding the crtE gene. This was done using alinker obtained using primer MUT11 (SEQ ID NO: 25) and MUT12 (SEQ ID NO:26). The wild type RBS was removed from pXI12-ZYIB-EINV4MUTRBS with NdeIand SpeI and the above mentioned linker was inserted. In the constructpXI12-ZYIB-EINV4MUTRBS2C all Flavobacterium RBS's have been replaced bysynthetic RBS's of the consensus sequence AAAGGAGG-7-8N-ATG (see table2). E. coli TG1 cells transformed with this construct showed that alsothis last RBS replacement had not interferred

TABLE 2 Nucleotide sequences of the synthetic ribosome binding sites inthe constructs pXI12-ZYIB3-EINV4MUTRBS2C, pXI12-ZYIB-EINV4MUTRBS2CCATand pXI12-ZYIB-EINV4 MUTRBS2CNEO. Nucleotides of the Shine-Dalgarnosequence preceding the indi- vidual carotenoid genes which arecomplementary to the 3′ ends of the 16S rRNA of B. subtilis are shown inbold. The 3′ ends of the 16S rRNA of E. coli is also shown ascomparison. The underlined AUG is the translation start site of thementioned gene. mRNA nucleotide sequence crtZ (SEQ ID NO: 48)AAAGGAGGGUUUCAUAUGAGC crtY (SEQ ID NO: 49) AAAGGAGGACACGUGAUGAGC crtI(SEQ ID NO: 50) AAAGGAGGCAAUUGAGAUGAGU crtB (SEQ ID NO: 51)AAAGGAGGAUCCAAUCAUGACC crtE (SEQ ID NO: 52) AAAGGAGGGUUUCUUAUGACG B.subtilis 16S rRNA (SEQ ID NO: 53) 3′-UCUUUCCUCCACUAG E. coli 16S rRNA(SEQ ID NO: 54) 3′- AUUCCUCCACUAG

with the ability to produce zeaxanthin. All the regions containing thenewly introduced synthetic RBS's were confirmed by sequencing. B.subtilis cells were transformed with plasmid pXI12-ZYIB-EINV4MUTRBS2 andone transformant having integrated the SFCO by reciprocal recombination,into the levan-sucrase gene of the chromosome, was selected. This strainwas named BS1012::SFCO2. Analysis of the carotenoid production of thisstrain show that the amounts zeaxanthin produced is approx. 40% of thezeaxanthin produced by E. coli cells transformed with the plasmid usedto get the B. subtilis transformant. Similar was the observation whencomparing the BS1012::SFCO1 strain with its E. coli counter part (30%).Although the E. coli cells have 18 times more carotenoid genes, thecarotenoid production is only a factor of 2-3 times higher. More drasticwas the difference observed in the carotenoid contents, between E. colicells carrying the pZea4 construct in about 200 copies and the E. colicarrying the plasmid pXI12-ZYIB-EINV4MUTRBS2C in 18 copies. The firsttransformant produced 48× more zeaxanthin than the latter one. Thisdifference seen can not only be attributed to the roughly 11 times morecarotenoid biosynthesis genes present in these transformants.Contributing to this difference is probably also the suboptimalperformance of the newly constructed SFCO, in which the overlappinggenes of the wild type Flavobacterium operon were separated to introducethe synthetic RBS's. This could have resulted in a lower translationefficiency of the rebuild synthetic operon (e.g. due to elimination ofputative translational coupling effects, present in the wild typeoperon).

In order to increase the carotenoid production, two new constructs weremade, pXI12-ZYIB-EINV4MUTRBS2CNEO and pXI12-ZYIB-EINV4 MUTRBS2CCAT,which after the integration of the SFCO into the levan-sucrase site ofthe chromosome, generate strains with an amplifiable structure asdescribed by [Janniere et al., Gene 40, 47-55 (1985)]. PlasmidpXI12-ZYIB-EINV4MUTRBS2CNEO has been deposited on May 25, 1995 at theDSM-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH(Germany) under accession No. DSM 10013. Such amplifiable structures,when linked to a resistance marker (e.g chloramphenicol, neomycin,tetracycline), can be amplified to 20-50 copies per chromosome. Theamplifiable structure consist of the SFCO, the resistance gene and thepXI12 sequence, flanked by direct repeats of the sac-B 3′ gene (see FIG.22). New strains having elevated numbers of the SFCO could now beobtained by selecting for transformants with increased level ofresistance to the antibiotic. To construct plasmidpXI12-ZYIB-EINV4MUTRBS2CNEO, the neomycin resistance gene was isolatedfrom plasmid pBEST501 with PstI and SmaI and subcloned into the PstI andEcoO1091 sites of the pUC18 vector. The resulting construct was namedpUC18-Neo. To get the final construct, the PmeI-AatII fragment ofplasmid pXI12-ZYIB-EINV4MUTRBS2C was replaced with the SmaI-AatIIfragment of pUC18-Neo, containing the neomycin resistance gene. PlasmidpXI12-ZYIB-EINV4MUTRBS2CCAT was obtained as follows: the chloramphenicolresistance gene of pC194 was isolated by PCR using the primer pair cat3(SEQ ID NO: 17) and cat4 (SEQ ID NO: 18). The fragment was digested withEcoRI and AatII and subcloned into the EcoRI and AatII sites of pUC18.The resulting plasmid was named pUC18-CAT. The final vector was obtainedby replacing the PmeI-AatIl fragment of pXI12-ZYIB-EINV4MUTRBS2C withthe EcoRI-AatII fragment of pUC18-CAT, carrying the chloramphenicolresistance gene. FIG. 23 summarizes the different steps to obtainaforementioned constructs. Both plasmids were transformed into B.subtilis strain 1012, and transformants resulting from a Campbell-typeintegration were selected. Two strains BS1012::SFCONEO1 andBS1012::SFCOCAT1 were chosen for further amplification. Individualcolonies of both strains were independently amplified by growing them indifferent concentrations of antibiotics as described in the methodssection. For the cat gene carrying strain, the chloramphenicolconcentrations were 60, 80, 120 and 150 mg/ml. For the neo gene carryingstrain, the neomycin concentrations were 160 and 180 mg/ml. In bothstrains only strains with minor amplifications of the SFCO's wereobtained. In daughter strains generated from strain BS1012::SFCONEO1,the resistance to higher neomycin concentrations correlated with theincrease in the number of SFCO's in the chromosome and with higherlevels of carotenoids produced by these cells. A different result wasobtained with daughter strains obtained from strain BS1012::SFCOCAT1. Inthese strains an increase up to 150 mg chloramphenicol/ml resulted, asexpected, in a higher number of SFCO copies in the chromosome.

EXAMPLE 7

Construction of CrtW Containing Plasmids and Use for CarotenoidProduction

Polymerase chain reaction based gene synthesis: The nucleotide sequenceof the artificial crtW gene, encoding the β-carotene β-4-oxygenase ofAlcaligenes strain PC-1, was obtained by back translating the amino acidsequence outlined in [Misawa, 1995], using the BackTranslate program ofthe GCG Wisconsin Sequence Analysis Package, Version 8.0 (GeneticsComputer Group, Madison, Wis., USA) and a codon frequency referencetable of E. coli (supplied by the Bach Translate Program). The syntheticgene consisting of 726 nucleotides was constructed basically accordingto the method described by [Ye, 1992]. The sequence of the 12oligonucleotides (crtW1-crtW12) required for the synthesis are shown inFIG. 25 (SEQ ID NO: 28). Briefly, the long oligonucleotides weredesigned to have short overlaps of 15-20 bases, serving as primers forthe extension of the oligonucleotides. After four cycles a few copies ofthe full length gene should be present which is then amplified by thetwo terminal oligonucleotides crtW15 (SEQ ID NO: 55) and crtW26. Thesequences for these two short oligonucleotides are for the forwardprimer crtW15 (5′-TATATCTAGAcatatgTCCGGTCGTAAA CCGG-3′) and for thereverse primer crtW26 (SEQ ID NO: 56) (5′-TATAgaattccacgtgTCA AGCACGACCACCGGTTTTAC G-3′), where the sequences matching the DNA templates areunderlined. Small cap letters show the introduced restriction sites(NdeI for the forward primer and EcoRI and PmlI for the reverse primer)for the latter cloning into the pALTER-Ex2 expression vector.

Polymerase chain reaction: All twelve long oligonucleotides(crtW1-crtW12; 7 nM each) and both terminal primers (crtW15 and crtW26;0.1 mM each) were mixed and added to a PCR reaction mix containingExpand™ High Fidelity polymerase (Boehringer, Mannheim) (3.5 units) anddNTP's (100 mM each). The PCR reaction was run for 30 cycles with thefollowing profile: 94° C. for 1 min, 50° C. for 2 min and 72° C. for 3min. The PCR reaction was separated on a 1% agarose gel, and the band ofapprox. 700 bp was excised and purified using the glass beads method(Geneclean Kit, Bio101, Vista, Calif., USA). The fragment wassubsequently cloned into the SmaI site of plasmid pUC18, using theSure-Clone Kit (Pharmacia, Uppsala, Sweden). The sequence of theresulting crtW synthetic gene was verified by sequencing with theSequenase Kit Version 1.0 (United States Biochemical, Cleveland, Ohio,USA). The crtW gene constructed by this method was found to containminor errors, which were subsequently corrected by site-directedmutagenesis.

Construction of plasmids: Plasmid pBIIKS(+)-CARVEG-E (see also Example5) (FIG. 26) contains the carotenoid biosynthesis genes (crtE, crtB,crtY, crtI and crtZ) of the Gram (−) bacterium Flavobacterium sp. strainR1534 WT (ATCC 21588) [Pasamontes, 1995 #732] cloned into a modifiedpBluescript II KS(+) vector (Stratagene, La Jolla, USA) carrying site Iof the B. subtilis veg promoter [LeGrice, 1986 #806]. This constitutivepromoter has been shown to be functional in E. coli . Transformants ofE. coli strain TG1 carrying plasmid pBIIKS(+)-CARVEG-E synthesisezeaxanthin. Plasmid pALTER-Ex2-crtW was constructed by cloning theNdeI-EcoRI restricted fragment of the synthetic crtW gene into thecorresponding sites of plasmid pALTER-Ex2 (Promega, Madison, Wis.).Plasmid pALTER-Ex2 is a low copy plasmid with the p15a origin ofreplication, which allows it to be maintained with ColE1 vectors in thesame host. Plasmid pBIIKS-crtEBIYZW (FIG. 26) was obtained by cloningthe HindIII-PmlI fragment of pALTER-Ex2-crtW into the HindIII and theblunt end made MluI site obtained by a fill in reaction with Klenowenzyme, as described elsewhere in [Sambrook, 1989 #505]. Inactivation ofthe crtZ gene was done by deleting a 285 bp NsiI-NsiI fragment, followedby a fill in reaction and religation, resulting in plasmidpBIIKS-crtEBIY[DZ]W. Plasmid pBIIKS-crtEBIY[DZW] carrying thenon-functional genes crtW and crtZ, was constructed by digesting theplasmid pBIIKS-crtEBIY[DZ]W with NdeI and HpaI, and subsequent selfreligation of the plasmid after filling in the sites with Klenow enzyme.E. coli transformed with this plasmid had a yellow-orange colour due tothe accumulation of β-carotene. Plasmid pBIIKS-crtEBIYZ[DW] has atruncated crtW gene obtained by deleting the NdeI-HpaI fragment inplasmid pBIIKS-crtEBIYZW as outlined above. PlasmidspALTER-Ex2-crtEBIY[DZW] and pALTER-Ex2-crtEBIYZ[DW], were obtained byisolating the BamHI-XbaI fragment from pBIIKS-crtEBIY[DZW] andpBIIKS-crtEBIYZ[DW], respectively and cloning them into the BamHI andXbaI sites of pALTER-Ex2. The plasmid pBIIKS-crtW was constructed bydigesting pBIIKS-crtEBIYZW with NsiI and SacI, and self-religating theplasmid after recessing the DNA overhangs with Klenow enzyme. FIG. 27compiles the relevant inserts of all the plasmids used in this paper.

Carotenoid analysis: E. coli TG-1 transformants carrying the differentplasmid constructs were grown for 20 hours in Luria-Broth mediumsupplemented with antibiotics (ampicillin 100 mg/ml, tetracyclin 12.5mg/ml) in shake flasks at 37° C. and 220 rpm. Carotenoids were extractedfrom the cells with acetone. The acetone was removed in vacuo and theresidue was re dissolved in toluene. The coloured solutions weresubjected to high-performance liquid chromatography (HPLC) analysiswhich was performed on a Hewlett-Packard series 1050 instrument. Thecarotenoids were separated on a silica column Nucleosil Si-100, 200×4mm, 3m. The solvent system included two solvents: hexane (A) andhexane/THF, 1:1 (B). A linear gradient was applied running from 13 to50% (B) within 15 minutes. The flow rate was 1.5 ml/min. Peaks weredetected at 450 nm by a photo diode array detector. The individualcarotenoid pigments were identified by their absorption spectra andtypical retention times as compared to reference samples of chemicallypure carotenoids, prepared by chemical synthesis and characterised byNMR, MS and UV-Spectra. HPLC analysis of the pigments isolated from E.coli cells transformed with plasmid pBIIKS-crtEBIYZW, carrying besidesthe carotenoid biosynthesis genes of Flavobacterium sp. strain R1534,also the crtW gene encoding the β-carotene ketolase of Alcaligenes PC-1[Misawa, 1995 #670] gave the following major peaks identified as:b-cryptoxanthin, astaxanthin, adonixanthin and zeaxanthin, based on theretention times and on the comparison of the absorbance spectra to givenreference samples of chemically pure carotenoids. The relative amount(area percent) of the accumulated pigment in the E. coli transformantcarrying pBIIKS-crtEBIYZW is shown in Table 3 [“CRX”: cryptoxanthin;“ASX”: astaxanthin; “ADX”: adonixanthin; “ZXN”: zeaxanthin; “ECM”:echinenone; “MECH”: 3-hydroxyechinenone, “CXN”: canthaxanthin]. The Σ ofthe peak areas of all identified carotenoids was defined as 100%.Numbers shown in Table 3 represent the average value of four independentcultures for each transformant. In contrast to the aforementionedresults, E. coli transformants carrying the same genes but on twoplasmids namely, pBIIKS-crtEBIYZ[DW] and pALTER-Ex2-crtW, showed adrastical drop in adonixanthin and a complete lack of astaxanthinpigments (Table 3), whereas the relative amount of zeaxanthin (%) hadincreased. Echinenone, hydroxyechinenone and canthaxanthin levelsremained unchanged compared to the transformants carrying all the crtgenes on the same plasmid (pBIIKS-crtEBIYZDW). PlasmidpBIIKS-crtEBIYZ[DW] is a high copy plasmid carrying the functional genesof crtE, crtB, crtY, crtI, crtZ of Flavobacterium sp. strain R1534 and atruncated, non-functional version of the crtW gene, whereas thefunctional copy of the crtW gene is located on the low copy plasmidpALTER-Ex2-crtW. To analyze the effect of overexpression of the crtWgene with respect to the crtZ gene, E. coli cells were co-transformedwith plasmid pBIIKS-crtW carrying the crtW gene on the high copy plasmidpBIIKS-crtW and the low copy construct pALTER-Ex2-crtEBIYZ[DW], encodingthe Flavobacterium crt genes. Pigment analysis of these transformants byHPLC monitored the presence of β-carotene, cryptoxanthin, astaxanthin,adonixanthin, zeaxanthin, 3-hydroxyechine-none and minute traces ofechinenone and canthaxanthin (Table 3).

Transformants harbouring the crtW gene on the low copy plasmidpALTER-Ex2-crtW and the genes crtE, crtB, crtY and crtI on the high copyplasmid pBIIKS-crtEBIY[DZW] expressed only minor amounts ofcanthaxanthin (6%) but high levels of echinenone (94%), whereas cellscarrying the crtW gene on the high copy plasmid pBIIKS-crtW and theother crt genes on the low copy construct pALTER-Ex2-crtEBIY[DZW], had78.6% and 21.4% of echinenone and canthaxanthin, respectively (Table 3).

TABLE 3 plasmids CRX ASX ADX ZXN ECH HECH CXN pBIIKS-crtEBIYZW 1.1 2.044.2 52.4 <1 <1 <1 pBIIKS-crtEBIYZ[W] + 2.2 — 25.4 72.4 <1 <1 <1pALTER-Ex2-crtW pBIIKS-crtEBIY[Z]W — — — — 66.5 — 33.5pBIIKS-crtEBIY[ZW] + pBIIKS- — — — — 94 — 6 crtW

EXAMPLE 8

Selective Carotenoid Production by Using the CrtW and CrtZ Genes of theGram Negative Bacterium E-396

In this section we describe E. coli transformants which accumulate onlyone (canthaxanthin) or two main carotenoids (astaxanthin, adonixanthin)and minor amounts of adonirubin, rather than the complex variety ofcarotenoids seen in most carotenoid producing bacteria [Yokoyama et al.,Biosci. Biotechnol. Biochem. 58:1842-1844 (1994)] and some of the E.coli transformants shown in Table 3. The ability to construct strainsproducing only one carotenoid is a major step towards a successfulbiotechnological carotenoid production process. This increase in theaccumulation of individual carotenoids accompanied by a decrease of theintermediates, was obtained by replacing the crtZ of FlavobacteriumR1534 and/or the synthetic crtW gene (see example 5) by their homologousgenes originating from the astaxanthin producing Gram negative bacteriumE-396 (FERM BP-4283) [Tsubokura et al., EP-application 0 635 576 A1].Both genes, crtW_(E396) and crtZ_(E396), were isolated and used toconstruct new plasmids as outlined below.

Isolation of a putative fragment of the crtW gene of strain E-396 by thepolymerase chain reaction: Based on protein sequence comparison of thecrtW enzymes of Agrobacterium aurantiacum, Alcaligenes PC-1 (WO95/18220)[Misawa et al., J.Bacteriol. 177: 6575-6584 (1995)] and Haematococcuspluvialis [Kajiwara et al., Plant Mol. Biol. 29:343-352 (1995)][Lotan etal., FEBS letters, 364:125-128 (1995)], two regions named I and II,having high amino acid conservation and located approx. 140 amino acidsappart, were identified and chosen to design the degenerate PCR primersshown below. The N-terminal peptide HDAMHG (region I) was used to designthe two 17-mer degenerate primer sequences crtW100 (SEQ ID NO: 57) andcrtW101 (SEQ ID NO: 58):

(SEQ ID NO: 57) crtW100: 5′-CA(C/T)GA(C/T)GC(A/C)ATGCA(C/T)GG-3′ (SEQ IDNO: 58) crtW101: 5′-CA(C/T)GA(C/T)GC(G/T)ATGCA(C/T)GG-3′

The C-terminal peptide H(W/H)EHH(R/L) corresponding to region II wasused design the two 17-mer degenerate primer with the antisensesequences crtW105 (SEQ ID NO: 59) and crtW106 (SEQ ID NO: 60):

(SEQ ID NO: 59) crtW105: 5′-AG(G/A)TG(G/A)TG(T/C)TC(G/A)TG(G/A)TG3′ (SEQID NO: 60) crtW106: 5′-AG(G/A)TG(G/A)TG(T/C)TCCCA(G/A)TG-3′

Polymerase chain reaction: PCR was performed using the GeneAmp Kit(Perkin Elmer Cetus) according to the manufacturer's instructions. Thedifferent PCR reactions contained combinations of the degenerate primers(crtW100/crtW105 or crtW100/crtW106 or crtW100/crtW105 orcrtW101/crtW106) at a final concentration of 50 pM each, together withgenomic DNA of the bacterium E-396 (200 ng) and 2.5 units of Taqpolymerase. In total 35 cycles of PCR were performed with the followingcycle profile: 95° C. for 30 sec, 55° C. for 30 sec, 72° C. for 30 sec.PCR reactions made with the following primer combinationscrtW100/crtW105 and crtW101/crtW105 gave PCR amplification products ofapprox. 500 bp which were in accordance with the expected fragment size.The 500 bp fragment, JAPclone8, obtained in the PCR reaction usingprimers crtW101 (SEQ ID NO: 58) and crtW105 (SEQ ID NO: 59) was excisedfrom an 1.5% agarose gel and purified using the GENECLEAN Kit andsubsequently cloned into the SmaI site of pUC18 using the Sure-CloneKit, according to the manufacturer's instructions. The resulting plasmidwas named pUC18-JAPclone8 and the insert was sequenced. Comparison ofthe determined sequence to the crtW gene of Agrobacterium aurantiacum(GenBank accession n° D58420) published by Misawa et al. in 1995(WO95/18220) showed 96% identity at the nucleotide sequence level,indicating that both organisms might be closely related.

Isolation of the crt cluster of the strain E-396: Genomic DNA of E-396was digested overnight with different combinations of restrictionsenzymes and separated by agarose gel electrophoresis before transferringthe resulting fragments by Southern blotting onto a nitrocellulosemembrane. The blot was hybridised with a ³²P labelled 334 bp fragmentobtained by digesting the aforementioned PCR fragment JAPclone8 withBssHII and MluI. An approx. 9,4 kb EcoRI/BamHI fragment hybridizing tothe probe was identified as the most appropiate for cloning since it islong enough to potentially carry the complete crt cluster. The fragmentwas isolated and cloned into the EcoRI and BamHI sites ofpBluescriptIIKS resulting in plasmid pJAPCL544 (FIG. 29). Based on thesequence of the PCR fragment JAPclone8, two primers were synthesized toobtain more sequence information left and right hand of this fragment.FIG. 30 shows the sequence obtained containing the crtW_(E396) (fromnucleotide 40 to 768) and crtZ_(E396) (SEQ ID NO: 33) (from nucleotide765 to 1253) genes of the bacterium E-396. The nucleotide sequence ofthe crtW_(E396) (SEQ ID NO: 30) gene is shown in FIG. 31 (SEQ ID NO: 31)and the encoded amino acid sequence in FIG. 32 (SEQ ID NO: 32). Thenucleotide sequence of the crtZ_(E396) gene is shown in FIG. 33 (SEQ IDNO: 33) and the corresponding amino acid sequence in FIG. 34 (SEQ ID NO:34). Comparison to the crtW_(E396) gene of E-396 to the crtW gene of A.aurantiacum showed 97% identity at the nucleotide level and 99% identityat the amino acid level. For the crtZ gene the values were 98% and 99%,respectively.

Construction of plasmids: Both genes, crtW_(E396) and crtZ_(E396), whichare adjacent in the genome of E-396, were isolated by PCR using primercrtW107 and crtW108 and the ExpandTM High Fidelity PCR system ofBoehringer Mannheim, according to the manufacturer's recommendations. Tofacilitate the subsequent cloning steps (see section below) the primercrt107 (SEQ ID NO: 61) (5′-ATCATATGAGCGCACATGCCCTGCCCAAGGC-3′) containsan artificial NdeI site (underlined sequence) spanning the ATG startcodon of the crtW_(E396) gene and the reverse primer crtW108 (SEQ ID NO:62) (5′-ATCTCGAGTCACGTGCGC TCCTGCGCCTCGGCC-3′) has an XhoI site(underlined sequence) just downstream of the TGA stop codon of thecrtZ_(E396) gene. The final PCR reaction mix had 10 pM of each primer,2.5 mg genomic DNA of the bacterium E-396 and 3.5 units of theTaqDNA/Pwo DNA polymerase mix. In total 35 cycles were performed withthe following cycle profile: 95° C., 1 min; 60° C., 1 min; 72° C. 1 min30 sec. The PCR product of approx. 1250 bp was isolated from the 1%agarose gel and purified using GENECLEAN before ligation into the SmaIsite pUC18 using the Sure-Clone Kit. The resulting construct was namedpUC18-E396crtWZPCR (FIG. 35). The functionality of both genes was testedas follows. The crtW_(E396) and crtZ_(E396) gene were isolated fromplasmid pUC18-E396crtWZPCR with NdeI and XhoI and cloned into the NdeIand SalI site of plasmid pBIIKS-crtEBIYZW resulting in plasmidpBIIKS-crtEBIY[E396WZ] (FIG. 36). E. coli TG1 cells transformed withthis plasmid produced astaxanthin, adonixanthin and adonirubin but nozeaxanthin (Table 4).

Plasmid pBIIKS-crtEBIY[E396W]DZ has a truncated non-functional crtZgene. FIG. 37 outlines the construction of this plasmid. The PCRreaction was run as outlined elsewhere in the text using primers crtW113(SEQ ID NO: 63)/crtW114 (SEQ ID NO: 64) and 200 ng of plasmidpUC18-JAPclone8 as template using 20 cycles with the following protocol:95° C., 45 sec/62° C., 20 sec/72° C., 20 sec)

primer crtW113: (SEQ ID NO: 63)(5′-ATATACATATGGTGTCCCCCTTGGTGCGGGTGC-3′) primer crtW114: (SEQ ID NO:64) (5′-TATGGATCCGACGCGTTCCCGGACCGCCACAATGC-3′)

The resulting 150 bp fragment was digested with BamHI and NdeI andcloned into the corresponding sites of pBIISK(+)-PCRRBScrtZ resulting inthe construct pBIISK(+)-PCRRBScrtZ-2. The final plasmid carrying thegenes crtE, crtB, crtI, crtY of Flavobacterium, the crtW_(E396) gene ofE-396 and a truncated non-functional crtZ gene of Flavobacterium wasobtained by isolating the MluI/NruI fragment (280 bp) ofpBIISK(+)-PCRRBScrtZ-2 and cloning it, into the MluI/PmlI sites ofplasmid pBIIKS-crtEBIY[E396WZ]. E. coli cells transformed with thisplasmid produced 100% canthaxanthin (Table 4; “CRX”: cryptoxanthin;“ASX”: astaxanthin; “ADX”: adonixanthin; “ZXN”: zeaxanthin; “ECH”:echinenone; “HECH”: 3-hydroxyechinenone; “CXN”: canthaxanthin; “BCA”:β-carotene; “ADR”: adonirubin; Numbers indicate the % of the individualcarotenoid of the total carotenoids produced in the cell.).

TABLE 4 plasmid CRX ASX ADX ZXN ECH HECH CXN BCA ADR pBIIKScrtEBIYZW 1.12.0 44.2 52.4 <1 <1 <1 pBIIKS- 74.4 19.8 5.8 crtEBIY[E396WZ] pBIIKS- 100crtEBIY[E396W]DZ

The results of E. coli transformants carrying pBIIKScrtEBIYZW (seeexample 7) are also shown in Table 4 to indicate the dramatic effect ofthe new genes crtW_(E396) and crtZ_(E396) on the carotenoids produced inthese new transformants.

EXAMPLE 9

Cloning of the Remaining Crt Genes of the Gram Negative Bacterium E-396

TG1 E. coli transformants carrying the pJAPCL544 plasmid did not producedetectable quantities of carotenoids (results not shown). Sequenceanalysis and comparison of the 3′ (BamHI site) of the insert of plasmidpJAPCL544, to the crt cluster of Flavobacterium R1534 showed that onlypart of the C-terminus of the crtE gene was present. This resultexplained the lack of carotenoid production in the aforementionedtransformants. To isolate the missing N-terminal part of the gene,genomic DNA of E-396 was digested by 6 restrictions enzymes in differentcombinations: EcoRI, BamHI, PstI, SacI, SphI and XbaI and transferred bythe Southern blot technique to nitrocellulose. Hybridization of thismembrane with the ³²P radio-labelled probe (a 463 bp PstI-BamHI fragmentoriginating from the 3′ end of the insert of pJAPCL544 (FIG. 29)highlighted a ˜1300 bp-long PstI-PstI fragment. This fragment wasisolated and cloned into the PstI site of pBSIIKS(+) resulting inplasmid pBSIIKS-#1296. The sequence of the insert is shown in FIG. 38(SEQ ID NO: 35) (small cap letters refer to new sequence obtained.Capital letters show the sequence also present in the 3′ of the insertof plasmid pJAPCL544). The complete crtE gene has therefore a length of882 bp (see FIG. 39) and encodes a GGPP synthase of 294 amino acids(FIG. 40) (SEQ ID NO: 37). The crtE enzyme has 38% identity with thecrtE amino acid sequence of Erwinia herbicola and 66% withFlavobacterium R1534 WT.

Construction of plasmids: To have a plasmid carrying the complete crtcluster of E-396, the 4.7 kb MluI/BamHI fragment encoding the genescrtW, crtZ, crtY, crtI and crtB was isolated from pJAPCL544 and clonedinto the MluI/BamHI sites of pUC18-E396crtWZPCR (see example 8). The newconstruct was named pE396CARcrtW-B (FIG. 41) and lacked the N-terminusof the crtE gene. The missing C-terminal part of the crtE gene was thenintroduced by ligation of the aforementioned PstI fragment ofpBIIKS-#1296 between the PstI sites of pE396CARcrtW-B. The resultingplasmid was named pE396CARcrtW-E (FIG. 41). The carotenoid distributionof the E. coli transformants carrying aforementioned plasmid were:adonixanthin (65%), astaxanthin (8%) and zeaxanthin (3%). The %indicated reflects the proportion of the total amount of carotenoidproduced in the cell.

EXAMPLE 10

Astaxanthin and adonixanthin production in Flavobacterium R1534

Among bacteria Flavobacterium may represent the best source for thedevelopment of a fermentative production process for 3R, 3R′ zeaxanthin.Derivatives of Flavobacterium sp. strain R1534, obtained by classicalmutagenesis have attracted in the past two decades wide interest for thedevelopment of a large scale fermentative production of zeaxanthin,although with little success. Cloning of the carotenoid biosynthesisgenes of this organism, as outlined in example 2, may allow replacementof the classical mutagenesis approach by a more rational one, usingmolecular tools to amplify the copy number of relevant genes, deregulatetheir expression and eliminate bottlenecks in the carotenoidbiosynthesis pathway. Furthermore, the introduction of additionalheterologous genes (e.g. crtW) will result in the production ofcarotenoids normally not synthesised by this bacterium (astaxanthin,adonirubin, adonixanthin, canthaxanthin, echinenone). The constructionof such recombinant Flavobacterium R1534 strains producing astaxanthinand adonixanthin will be outlined below.

Gene Transfer into Flavobacterium sp

Plasmid transfer by conjugative mobilization: For the conjugationalcrosses we constructed plasmid pRSF1010-Amp^(r), a derivative of thesmall (8.9 kb) broad host range plasmid RSF1010 (IncQ incompatibilitygroup) [Guerry et al., J. Bacteriol. 117:619-630 (1974)] and used E.coli S17-1 as the mobilizing strain [Priefer et al., J. Bacteriol.163:324-330 (1985)]. In general any of the IncQ plasmids (e.g. RSF1010,R300B, R1162) may be mobilized into rifampicin resistant Flavobacteriumif the transfer functions are provided by plasmids of the IncP1 group(e.g. R1, R751).

Rifampicin resistant (Rif^(r)) Flavobacterium R1534 cells were obtainedby selection on 100 mg rifampicin/ml. One resistant colony was pickedand a stock culture was made. The conjugation protocol was as follows:

Day 1

grow 3 ml culture of Flavobacterium R1534 Rif^(r) for 24 hours at 30° C.in Flavobacter medium (F-medium) (see example 1)

grow 3 ml mobilizing E. coli strain carrying the mobilizable plasmid O/Nat 37° C. in LB medium. (e.g E. coli S17-1 carrying pRSF1010-Amp^(r) orE. coli TG-1 cells carrying R751 and pRSF1010-Amp^(r))

Day 2

pellet 1 ml of the Flavobacterium R1534 Rif^(r) cells and resuspend in1ml of fresh F-medium.

pellet 1 ml of E. coli cells (see above) and resuspend in 1 ml of LBmedium.

donor and recipient cells are then mixed in a ratio of 1:1 and 1:10 inan Eppendorf tube and 30 ml are then applied onto a nitrocellulosefilter plated on agar plates containing F-medium and incubated O/N at30° C.

Day 3

the conjugational mixtures were washed off with F-medium and plated onF-medium containing 100 mg rifampicin and 100 mg ampicillin/ml forselection of transconjugants and inhibition of the donor cells.

Day 6-8

Arising clones are plated once more on F-medium containing 100 mgRif and100 mg Amp/ml before analysis.

Plasmid transfer by electroporation: The protocol for the eletroporationis as follows:

1. add 10 ml of O/N culture of Flavobacterium sp. R1534 into 500mlF-medium and incubate at 30° C. until OD600=0.8-0.1

2. harvest cells by centrifugation at 4000 g at 4° C. for 10 min.

3. wash cells in equal volume of ice-cold deionized water (2 times)

4. resuspend bacterial pellet in 1 ml ice-cold deionized water

5. take 50 ml of cells for electroporation with 0.1 mg of plasmid DNA

6. electroporation was done using field strengths between 15 and 25kV/cm and 1-3 ms.

7. after electroporation cells were immediately diluted in 1 ml ofF-medium and incubated for 2 hours at 30° C. at 180 rpm before platingon F-medium plates containing the respective selective antibioticum.

Plasmid constructions: Plasmid pRSF101-Amp^(r) was obtained by cloningthe Amp^(r) gene of pBR322 between the EcoRI/NotI sites of RSF1010. TheAmp^(r) gene originates from pBR322 and was isolated by PCR usingprimers AmpR1 (SEQ ID NO: 65) and AmpR2 (SEQ ID NO: 66) as shown in FIG.42.

AmpR1 (SEQ ID NO: 65)

5′-TATATCGGCCGACTAGTAAGCTTCAAAAAGGATCTTCACCTAG-3′ the underlinedsequence contains the introduced restriction sites for EagI, SpeI andHindIII to facilitate subsequent constructions.

AmpR2 (SEQ ID NO: 66)

5′-ATATGAATTCAATAATATTGAAAAAGGAAG-3′ the underlined sequence correspondsto an introduced EcoRI restriction site to facilitate cloning intoRSF1010 (see FIG. 42).

The PCR reaction mix had 10 pM of each primer (AmpR1 (SEQ ID NO:65)/AmpR2 (SEQ ID NO: 66)), 0.5 mg plasmid pBR322 and 3.5 units of theTaqDNA/Pwo DNA polymerase mix. In total 35 amplification cycles weremade with the profile: 95° C., 45 sec; 59° C., 45 sec, 72° C., 1 min.The PCR product of approx. 950 was extracted once with phenol/chloroformand precipitated with 0.3 M NaAcetate and 2 vol. Ethanol. The pellet wasresuspended in H₂O and digested with EcoRI and EagI O/N. The digestionwas separated by electrophoresis and the fragment isolated from the 1%agarose gel and purified using GENECLEAN before ligation into the EcoRIand NotI sites of RSF1010. The resulting plasmid was namedpRSF1010-Amp^(r)(FIG. 42).

Plasmid RSF1010-Amprcrt1 was obtained by isolating the HindIII/NotIfragment of pBIIKS-crtEBIY[E396WZ] and cloning it between theHindIII/EagI sites of RSF1010-Amp^(r) (FIG. 43). The resulting plasmidRSF1010-Ampr-crt1 carries crtW_(E396), crtZ_(E396), crtY genes and theN-terminus of the crtI gene (non-functional). Plasmid RSF1010-Ampr-crt2carrying a complete crt cluster composed of the genes crtW_(E396) andcrtZ_(E396) of E-396 and the crtY, crtI, crtB and crtE of FlavobacteriumR1534 was obtained by isolating the large HindIII/XbaI fragment ofpBIIKS-crtEBIY[E396WZ] and cloning it into the SpeI/HindIII sites ofRSF1010-Amp^(r) (FIG. 43).

Flavobacterium R1534 transformants carrying either plasmidRSF1010-Amp^(r), Plasmid RSF1010-Amp^(r)-crt1 or PlasmidRSF1010-Amp^(r)-crt2 were obtained by conjugation as outlined aboveusing E. coli S17-1 as mobilizing strain.

Comparison of the carotenoid production of two flavobacteriumtransformants: Overnight cultures of the individual transformants werediluted into 20 ml fresh F-medium to have a final starting OD600 of 0.4.Cells were harvested after growing for 48 hours at 30° C. and carotenoidcontents were analysed as outlined in example 7. Table 5 shows theresult of the three control cultures Flavobacterium [R1534 WT], [R1534WT RifR] (rifampicin resistant) and [R1534WT Rifr RSF1010-AmpR] (carriesthe RSF1010-Amp^(r) plasmid) and the two transformants [R1534 WTRSF1010-AmpR-crt1] and [R1534 WT RSF1010-AmpR-crt2]. Both lattertransformants are able to synthesise astaxanthin and adonixanthin butlittle zeaxanthin. Most interesting is the [R1534 WT RSF1010-AmpR-crt2]Flavobacterium transformant which produces approx. 4 times morecarotenoids than the R1534 WT. This increase in total carotenoidproduction is most likely due to the increase of the number ofcarotenoid biosynthesis clusters present in these cell (e.g. correspondsto the total copy number of plasmids in the cell).

TABLE 5 total carotenoid carotenoids % of total content in %Transformant dry weight of dry weight R1534 WT 0.039% b-Carotin 0.001%0.06% b-Cryptoxanthin 0.018% Zeaxanthin R1534 Rifr 0.036% b-Carotin0.002% 0.06% b-Cryptoxanthin 0.022% Zeaxanthin R1534 Rifr 0.021%b-Carotin 0.002% 0.065% [RSF1010-Ampr] b-Cryptoxanthin 0.032% ZeaxanthinR1534 Rifr 0.022% Astaxanthin 0.075% 0.1% [RSF1010-Ampr-crt1]Adonixanthin 0.004% Zeaxanthin R1534 Rifr 0.132% b-Carotin 0.006% 0.235%[RSF1010-Ampr-crt2] Echinenon 0.004% Hydroxyechinenon 0.003%b-Cryptoxanthin 0.044% Astaxanthin 0.039% Adonixanthin 0.007% Zeaxanthin

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 66 <210> SEQ ID NO 1 <211> LENGTH: 8625<212> TYPE: DNA <213> ORGANISM: Flavobacterium sp. R1534 <220> FEATURE:<221> NAME/KEY: unsure <222> LOCATION: (8348)..(8349)<221> NAME/KEY: unsure <222> LOCATION: (8539)..(8540)<221> NAME/KEY: unsure <222> LOCATION: (8581) <221> NAME/KEY: unsure<222> LOCATION: (8590) <221> NAME/KEY: unsure <222> LOCATION: (8592)<221> NAME/KEY: unsure <222> LOCATION: (8602)..(8604) <400> SEQUENCE: 1ggatccgcgc ctggccgttc gcgatcagca gccgcccttg cggatcggtc ag#catcatcc     60ccatgaaccg cagcgcacga cgcagcgcgc gccccagatc gggcgcgtcc ag#cacggcat    120gcgccatcat cgcgaaggcc cccggcggca tggggcgcgt gcccattccg aa#gaactcgc    180agcctgtccg ctgcgcaagg tcgcgccaga tcgcgccgta ttccgatgca gt#gacgggcc    240cgatgcgcgt gggcccgccc tgccccgccg ccaccagcgc atcgcgcacg aa#cccttccg    300agatgatgtg ctgatccatg gcccgtcatt gcaaaaccga tcaccgatcc tg#tcgcgtga    360tggcattgtt tgcaatgccc cgagggctag gatggcgcga aggatcaagg gg#gggagaga    420catggaaatc gagggacggg tctttgtcgt cacgggcgcc gcatcgggtc tg#ggggcggc    480ctcggcgcgg atgctggccc aaggcggcgc gaaggtcgtg ctggccgatc tg#gcggaacc    540gaaggacgcg cccgaaggcg cggttcacgc ggcctgcgac gtgaccgacg cg#accgctgc    600gcagacggcc atcgcgctgg cgaccgaccg cttcggcagg ctggacggcc tt#gtgaactg    660cgcgggcatc gcgccggccg aacggatgct gggccgcgac gggccgcatg ga#ctggacag    720ctttgcccgt gcggtcacga tcaacctgat cggcagcttc aacatggccc gc#cttgcagc    780cgaggcgatg gcccggaacg agcccgtccg gggcgagcgt ggcgtgatcg tc#aacacggc    840ctcgatcgcg gcgcaggacg gacagatcgg acaggtcgcc tatgcggcca gc#aaggcggg    900cgtggcgggc atgacgctgc cgatggcccg cgaccttgcg cggcacggca tc#cgcgtcat    960gaccatcgcg cccggcatct tccgcacccc gatgctggag gggctgccgc ag#gacgttca   1020ggacagcctg ggcgcggcgg tgcccttccc ctcgcggctg ggagagccgt cg#gaatacgc   1080ggcgctgttg caccacatca tcgcgaaccc catgctgaac ggagaggtca tc#cgcctcga   1140cggcgcattg cgcatggccc ccaagtgaag gagcgtttca tggaccccat cg#tcatcacc   1200ggcgcgatgc gcaccccgat gggggcattc cagggcgatc ttgccgcgat gg#atgccccg   1260acccttggcg cggacgcgat ccgcgccgcg ctgaacggcc tgtcgcccga ca#tggtggac   1320gaggtgctga tgggctgcgt cctcgccgcg ggccagggtc aggcaccggc ac#gtcaggcg   1380gcgcttggcg ccggactgcc gctgtcgacg ggcacgacca ccatcaacga ga#tgtgcgga   1440tcgggcatga aggccgcgat gctgggccat gacctgatcg ccgcgggatc gg#cgggcatc   1500gtcgtcgccg gcgggatgga gagcatgtcg aacgccccct acctgctgcc ca#aggcgcgg   1560tcggggatgc gcatgggcca tgaccgtgtg ctggatcaca tgttcctcga cg#ggttggag   1620gacgcctatg acaagggccg cctgatgggc accttcgccg aggattgcgc cg#gcgatcac   1680ggtttcaccc gcgaggcgca ggacgactat gcgctgacca gcctggcccg cg#cgcaggac   1740gccatcgcca gcggtgcctt cgccgccgag atcgcgcccg tgaccgtcac gg#cacgcaag   1800gtgcagacca ccgtcgatac cgacgagatg cccggcaagg cccgccccga ga#agatcccc   1860catctgaagc ccgccttccg tgacggtggc acggtcacgg cggcgaacag ct#cgtcgatc   1920tcggacgggg cggcggcgct ggtgatgatg cgccagtcgc aggccgagaa gc#tgggcctg   1980acgccgatcg cgcggatcat cggtcatgcg acccatgccg accgtcccgg cc#tgttcccg   2040acggccccca tcggcgcgat gcgcaagctg ctggaccgca cggacacccg cc#ttggcgat   2100tacgacctgt tcgaggtgaa cgaggcattc gccgtcgtcg ccatgatcgc ga#tgaaggag   2160cttggcctgc cacacgatgc cacgaacatc aacggcgggg cctgcgcgct tg#ggcatccc   2220atcggcgcgt cgggggcgcg gatcatggtc acgctgctga acgcgatggc gg#cgcggggc   2280gcgacgcgcg gggccgcatc cgtctgcatc ggcgggggcg aggcgacggc ca#tcgcgctg   2340gaacggctga gctaattcat ttgcgcgaat ccgcgttttt cgtgcacgat gg#gggaaccg   2400gaaacggcca cgcctgttgt ggttgcgtcg acctgtcttc gggccatgcc cg#tgacgcga   2460tgtggcaggc gcatggggcg ttgccgatcc ggtcgcatga ctgacgcaac ga#aggcaccg   2520atgacgccca agcagcaatt ccccctacgc gatctggtcg agatcaggct gg#cgcagatc   2580tcgggccagt tcggcgtggt ctcggccccg ctcggcgcgg ccatgagcga tg#ccgccctg   2640tcccccggca aacgctttcg cgccgtgctg atgctgatgg tcgccgaaag ct#cgggcggg   2700gtctgcgatg cgatggtcga tgccgcctgc gcggtcgaga tggtccatgc cg#catcgctg   2760atcttcgacg acatgccctg catggacgat gccaggaccc gtcgcggtca gc#ccgccacc   2820catgtcgccc atggcgaggg gcgcgcggtg cttgcgggca tcgccctgat ca#ccgaggcc   2880atgcggattt tgggcgaggc gcgcggcgcg acgccggatc agcgcgcaag gc#tggtcgca   2940tccatgtcgc gcgcgatggg accggtgggg ctgtgcgcag ggcaggatct gg#acctgcac   3000gcccccaagg acgccgccgg gatcgaacgt gaacaggacc tcaagaccgg cg#tgctgttc   3060gtcgcgggcc tcgagatgct gtccattatt aagggtctgg acaaggccga ga#ccgagcag   3120ctcatggcct tcgggcgtca gcttggtcgg gtcttccagt cctatgacga cc#tgctggac   3180gtgatcggcg acaaggccag caccggcaag gatacggcgc gcgacaccgc cg#cccccggc   3240ccaaagggcg gcctgatggc ggtcggacag atgggcgacg tggcgcagca tt#accgcgcc   3300agccgcgcgc aactggacga gctgatgcgc acccggctgt tccgcggggg gc#agatcgcg   3360gacctgctgg cccgcgtgct gccgcatgac atccgccgca gcgcctaggc gc#gcggtcgg   3420gtccacaggc cgtcgcggct gatttcgccg ccgcgcaggc gcgatgcggc cg#cgtccaag   3480cctccgcgcg ccagaagccc gatcttggca gccttcgacg tgctgatccg ct#ggcgatag   3540gcctcggggc caccctgccg gatgcgcgtc ccgattgcgc gatagatacg ca#gcgcggcg   3600gcgatcgacc acgcgcagcg cggcggcaga tgcggaagcc cctgccgcgc cg#aggcataa   3660tagggctcgg ccgcgtcaag caggcggatg atgacggaat agagcgcgtc cg#aaggcacc   3720ggaccctcaa ccgtcgcccc cgcctcggcc agccagtcgg caggcagata gc#agcgcccg   3780atggcggcat cgtcgatcac gtcgcgagcg atgttcgtca gctggaacgc aa#ggcccaga   3840tcgcaggcgc gatccagcac cgcatcgtcc tgcacgccca tcacccgcgc ca#tcatcacg   3900cccacgaccc ccgcgacgtg gtaggaatat tccagcacgt catccaggct gc#ggtattcg   3960cgatccgcga catccatcgc gaaaccctcg atcaggtcca tcggccaaag gt#ccgggaaa   4020tcatgccgcc gggcgacctg gcgcagcgcc gcgaagggcg gcgacatcgg gc#cgtcctcg   4080tgcagcgcgg ccagcgtgtc ggcgcgcagc gcccccagcc gcgcctgtgg gt#cgccgccc   4140gcctcggggg cagaacccat cacctgcccg tcgatcacgt catccgcatg cc#tgcaccag   4200gcatagagca tgaccgtatc ctcgcggatg ccgggcggca tcagcttggc cg#cctgcgcg   4260aagctttgcg aaccctgcgc gatggccgct tcggaagtcg ccgtcagatc gg#tcatgcga   4320cggccaggtc cgacagcatg acctgcgccg tggccttggc gctgccaacg ac#acccggga   4380tgcccgcacc cggatgcgtg cccgccccca cgatgtagaa gttcgggatc gc#gcggtcgc   4440ggttatgcgg gcggaaccag gcggattgcg tcaggatcgg ctcgaccgag aa#ggcgctgc   4500cgtgatgggc cgacagttcg gtgctgaaat cggcggggct gaagatgcgg ct#gacggtca   4560ggtgcttgcg caggtcgggg atggcgcggc gctccagttc ctcgaagatg cg#ctcggcat   4620agcccggggc ctcggcttcc caatcgacat cggcgcggcc cagatgcgga ac#gggcgcaa   4680ggacgtaatg cgtggacatc ccctcggggg ccaggctggg atcggtcacg ca#gggcgaat   4740gcagatacat cgagaaatcg tccggcaggc gtggcccgtt gaagatctcg tt#caccagcc   4800ccttgtagcg cgggccgaag atgacgctgt ggtgggccag gttctcgggg cg#cttggaca   4860ggccgaaatg cagcacgaac agcgacatcg accagcgctg ccggttcagg at#cgcggcct   4920tggtgcgccc gcggcgggta tggcccagca ggtcgcgata gctgtgcatc ac#gtcgccgt   4980tgctggccac cgtatccgcg cgcaactgcc gcccgtccag cagcgtgacg cc#cgtggcgc   5040gatcgccctc ggtgtcgatc cgcgtgacgc gggcattcag cagcagcgtg cc#gccaagac   5100gctcgaacag ggcgaccatg cccgcgacca gctggttggt gccgcccttg gc#gaaccaga   5160cgccgccgcg ccgttccagc gcatggatca gcgcatagat cgagctggtc ga#aaacgggt   5220tcccgccgac cagcagcgtg tggaacgaga aggcctgccg cagatgcggg tc#ctggatga   5280agcgcgccac catgctgtgg accgagcggt atgcctgcag gcgcatcagc gc#cggcgcgg   5340cgttcagcat ctggcccagc ttcaggaagg gcgtggtccc cagcttcaga ta#cccctcgc   5400gatagacctc ctcggcgtaa tcgtggaagc ggcgatagcc atcgacatcg gc#gggattga   5460aggaggcgac ctggcggatc agctcgtcgt cgtcgttcac gtattcgaag ct#gcggccgt   5520ccgcccatgt cagccggtag aagggcgaga ccggcagcag cgtcacgtca cg#ctccatcg   5580gttggccgct gagggcccac agctctcgca ggctgtcggg gtcggtcacg ac#cgtcgggc   5640ctgcatcgaa gacgtggccc tgatcgttcc agacataggc gcggccgccg gg#cttgtcgc   5700gggcctcgac gatggtggtc gcgatgccgg ccgattgcag gcggatggca ag#cgcaagcc   5760cgccgaaacc tgcgccgatg acgatggcgg aactcatgct ctctcctgca gc#agggggcg   5820ttcgggcagg cagcgcacgg cctgcgacag cggaatgggc gggcgtccgg tg#acgatgcg   5880aagccggtcg gccaatgtca ggcgcccggc atagaagcgc tcgatcagcg gc#tgcggcag   5940gcggtagaac cgctgcagca ggcgatagcg acggtcgggc gggcagccgc gg#aacagcat   6000ccggttcagc agccgcagga agcggtcgcg atccgcgcga tcgatggccc ag#ccgcgcac   6060cgcgcgacgg gcggacgcgg tcgtcaggtc gcgcgccgcg atggcatccg cg#acctgcgc   6120ggcatagggc agcgaatatc cggtgacggg gtggaacagc cctgccccca gc#ccaaccgg   6180caccgccccc tgcgcgtggt cgcgccagaa gcctatggcg tcatgggcca gc#gcgatggg   6240caggatgccc ctttcgcgcc gcatctcctg cccggtccag ccccgcctgg cg#gcatagtc   6300cagcgacgcc tgcgccagcg cgccatcgtc cagatcgccg ccgtcgctgt ag#cgcgtatc   6360ctcgatcagg atgcgggtgg gactgaaggg cagcagatag atgaagcggt ac#ccgtccat   6420ctgcggaacg gtcgcgtcca tgatcatcgg gcgctcgacg ccatgggggg cg#tcggtctc   6480gatctcgacg cccacgaatt tctggaaacc cacggtcagg tgcggggtct cg#acggcacc   6540acgggcgtcg atcacgcagg cagcctcgat ccgcgagccg tccgtcagcg tc#gcgccggt   6600atcgtccagc gtcgcgacat gcgtattcca ccgcagatcg acaccctgca gc#agcccgat   6660cagcgcgccc gcctcgatcg agccatagcc tgtcgtcagg cggcgcgaat gg#tcgggaaa   6720cgcgacctcc tgatccgtcc attcgccgcg acgaatgggc gacaggcgcg cc#agccattc   6780gggcgaaaga tccgtgtcgt ggcaggacca ggtgtgctgg tccgaggggc cg#gaccgcgc   6840gtcgagcatc acgatgcgcg catccggtct gcggtcgcga acggcaagcg cg#atcagcgc   6900accggacagc cccgcgcccg cgatcagcag atcatggctc atgtattgcg at#ccgcccct   6960tcgcggtcct tcagcagcgc gcccgagcgt ttcagctctg ccttgaggct gt#cgaccgag   7020ggcgcccaga tgaaaccgaa gctgacgcag ttctcgcggc catggaccgc gt#gatgcatc   7080ctgtgtgcct ggtagacgcg acgaagatag ccgcgcttgg ggacatagcg ga#acggccag   7140cgcccatgca ccaagccgtc atgcaggaaa tagtagatca gcccgtagca gg#tgaccccc   7200accgccagcc accaggccag atccgacccc atcgcgccga tcgcgaacag ca#cgatcgag   7260attaccgcga agatgacgcc atagaggtcg ttcttctcga gcgcgtggtc gt#gatcctcg   7320tcgtggtgcg atttatgcca gccccagccc agggggccat gcatgatcca cc#gatggacg   7380gagtaggccg tcagctccat cgcggcgacg gtcaggatga cggtcaggat tg#cggcccaa   7440gtgctcatgc cggccccttg cttgatatga cagggaacag gctacgctgc cg#cgcggtgc   7500atgaccagcc catcggggtg cgaccaaagg gcatcgcgtg acatctgcgt tc#agggctca   7560taggcggatc atccgtgaca ttcgccgccg aacgcggcag gcgcatcacg cg#ttccgtcg   7620ctggaaatat taatgttttc ccgaagatgg tcggggcgag aggattcgaa cc#tccgacct   7680acggtaccca aaaccgtcgc gctaccaggc tgcgctacgc cccgactgcg ga#aggcttta   7740gccgattgtt ccggcaaggg aaagacctag tcgcaggcca ggaccgcatt gt#cgcccatg   7800cccggatgcg ccatcggctg accgggcttc aggccaaggc gatccgcctc tc#cgcccgcg   7860atttcgagga cgaacagccg gtcggggtcc ggatcgccga ccgccgcgcc cg#gaatgggc   7920gtctcgtcca gcgggcgcgc attgcggtgg atgtggcgga tgacgccggt tt#catccgca   7980aagaccatgt ccagcgggat cagtgtgttg cgcatccaga aggacaccgg ct#ggggcgat   8040tcgtagatga acagcattcc ggtgcccgca ggcagctcct tgcggaacat ca#ggccctgc   8100gcgcgctctt cggggctgtc cgcgacctcg acccgaaacc cgagcgtttc cg#caccggta   8160tcgacgacaa gactgccggg cgcgcattcc accgccgccg cggcggcggg ca#tcaggacc   8220gcaagaagcg ctgcggcctt actcggccac atgggcaaga taggactgct cg#gcgccgag   8280atcctgctga ccctgcgcat cctcgttccg gtcatgcagc gccaggtccc at#gccgcgat   8340ctgcgcgnnc atcagcccgc gcggaccctc gacgacgcgg aggcagatcg cc#tcgccgat   8400cacgaggtcc gagaagccgg aatgacggag cacctcgata tggatgaaca cg#tcctcggg   8460gtggccgaag atgttggcga accgggaaaa ggcccttggc cttgtcgaac ca#cttgacgc   8520gggccggacg cagcggcann cgtccagatg ctcgatcacc tcggcatcca ga#tcggcgat   8580 nggggggtgn cngtcgcttt cnnncggttc gatcgacagg acctc   #                8625 <210> SEQ ID NO 2 <211> LENGTH: 295<212> TYPE: PRT <213> ORGANISM: Flavobacterium sp. R1534<400> SEQUENCE: 2 Met Thr Pro Lys Gln Gln Phe Pro Leu Arg As#p Leu Val Glu Ile Arg   1               5  #                 10 #                 15 Leu Ala Gln Ile Ser Gly Gln Phe Gly Val Va#l Ser Ala Pro Leu Gly              20      #             25     #             30 Ala Ala Met Ser Asp Ala Ala Leu Ser Pro Gl#y Lys Arg Phe Arg Ala          35          #         40         #         45 Val Leu Met Leu Met Val Ala Glu Ser Ser Gl#y Gly Val Cys Asp Ala      50              #     55             #     60 Met Val Asp Ala Ala Cys Ala Val Glu Met Va#l His Ala Ala Ser Leu  65                  # 70                 # 75                  # 80 Ile Phe Asp Asp Met Pro Cys Met Asp Asp Al#a Arg Thr Arg Arg Gly                  85  #                 90 #                 95 Gln Pro Ala Thr His Val Ala His Gly Glu Gl#y Arg Ala Val Leu Ala             100       #           105      #           110 Gly Ile Ala Leu Ile Thr Glu Ala Met Arg Il#e Leu Gly Glu Ala Arg         115           #       120          #       125 Gly Ala Thr Pro Asp Gln Arg Ala Arg Leu Va#l Ala Ser Met Ser Arg     130               #   135              #   140 Ala Met Gly Pro Val Gly Leu Cys Ala Gly Gl#n Asp Leu Asp Leu His 145                 1 #50                 1#55                 1 #60 Ala Pro Lys Asp Ala Ala Gly Ile Glu Arg Gl#u Gln Asp Leu Lys Thr                 165   #               170  #               175 Gly Val Leu Phe Val Ala Gly Leu Glu Met Le#u Ser Ile Ile Lys Gly             180       #           185      #           190 Leu Asp Lys Ala Glu Thr Glu Gln Leu Met Al#a Phe Gly Arg Gln Leu         195           #       200          #       205 Gly Arg Val Phe Gln Ser Tyr Asp Asp Leu Le#u Asp Val Ile Gly Asp     210               #   215              #   220 Lys Ala Ser Thr Gly Lys Asp Thr Ala Arg As#p Thr Ala Ala Pro Gly 225                 2 #30                 2#35                 2 #40 Pro Lys Gly Gly Leu Met Ala Val Gly Gln Me#t Gly Asp Val Ala Gln                 245   #               250  #               255 His Tyr Arg Ala Ser Arg Ala Gln Leu Asp Gl#u Leu Met Arg Thr Arg             260       #           265      #           270 Leu Phe Arg Gly Gly Gln Ile Ala Asp Leu Le#u Ala Arg Val Leu Pro         275           #       280          #       285 His Asp Ile Arg Arg Ser Ala     290               #   295<210> SEQ ID NO 3 <211> LENGTH: 303 <212> TYPE: PRT<213> ORGANISM: Flavobacterium sp. R1534 <400> SEQUENCE: 3Met Thr Asp Leu Thr Ala Thr Ser Glu Ala Al #a Ile Ala Gln Gly Ser  1               5  #                 10  #                 15Gln Ser Phe Ala Gln Ala Ala Lys Leu Met Pr #o Pro Gly Ile Arg Glu             20      #             25      #             30Asp Thr Val Met Leu Tyr Ala Trp Cys Arg Hi #s Ala Asp Asp Val Ile         35          #         40          #         45Asp Gly Gln Val Met Gly Ser Ala Pro Glu Al #a Gly Gly Asp Pro Gln     50              #     55              #     60Ala Arg Leu Gly Ala Leu Arg Ala Asp Thr Le #u Ala Ala Leu His Glu 65                  # 70                  # 75                  # 80Asp Gly Pro Met Ser Pro Pro Phe Ala Ala Le #u Arg Gln Val Ala Arg                 85  #                 90  #                 95Arg His Asp Phe Pro Asp Leu Trp Pro Met As #p Leu Ile Glu Gly Phe            100       #           105       #           110Ala Met Asp Val Ala Asp Arg Glu Tyr Arg Se #r Leu Asp Asp Val Leu        115           #       120           #       125Glu Tyr Ser Tyr His Val Ala Gly Val Val Gl #y Val Met Met Ala Arg    130               #   135               #   140Val Met Gly Val Gln Asp Asp Ala Val Leu As #p Arg Ala Cys Asp Leu145                 1 #50                 1 #55                 1 #60Gly Leu Ala Phe Gln Leu Thr Asn Ile Ala Ar #g Asp Val Ile Asp Asp                165   #               170   #               175Ala Ala Ile Gly Arg Cys Tyr Leu Pro Ala As #p Trp Leu Ala Glu Ala            180       #           185       #           190Gly Ala Thr Val Glu Gly Pro Val Pro Ser As #p Ala Leu Tyr Ser Val        195           #       200           #       205Ile Ile Arg Leu Leu Asp Ala Ala Glu Pro Ty #r Tyr Ala Ser Ala Arg    210               #   215               #   220Gln Gly Leu Pro His Leu Pro Pro Arg Cys Al #a Trp Ser Ile Ala Ala225                 2 #30                 2 #35                 2 #40Ala Leu Arg Ile Tyr Arg Ala Ile Gly Thr Ar #g Ile Arg Gln Gly Gly                245   #               250   #               255Pro Glu Ala Tyr Arg Gln Arg Ile Ser Thr Se #r Lys Ala Ala Lys Ile            260       #           265       #           270Gly Leu Leu Ala Arg Gly Gly Leu Asp Ala Al #a Ala Ser Arg Leu Arg        275           #       280           #       285Gly Gly Glu Ile Ser Arg Asp Gly Leu Trp Th #r Arg Pro Arg Ala    290               #   295               #   300 <210> SEQ ID NO 4<211> LENGTH: 494 <212> TYPE: PRT<213> ORGANISM: Flavobacterium sp. R1534 <400> SEQUENCE: 4Met Ser Ser Ala Ile Val Ile Gly Ala Gly Ph #e Gly Gly Leu Ala Leu  1               5  #                 10  #                 15Ala Ile Arg Leu Gln Ser Ala Gly Ile Ala Th #r Thr Ile Val Glu Ala             20      #             25      #             30Arg Asp Lys Pro Gly Gly Arg Ala Tyr Val Tr #p Asn Asp Gln Gly His         35          #         40          #         45Val Phe Asp Ala Gly Pro Thr Val Val Thr As #p Pro Asp Ser Leu Arg     50              #     55              #     60Glu Leu Trp Ala Leu Ser Gly Gln Pro Met Gl #u Arg Asp Val Thr Leu 65                  # 70                  # 75                  # 80Leu Pro Val Ser Pro Phe Tyr Arg Leu Thr Tr #p Ala Asp Gly Arg Ser                 85  #                 90  #                 95Phe Glu Tyr Val Asn Asp Asp Asp Glu Leu Il #e Arg Gln Val Ala Ser            100       #           105       #           110Phe Asn Pro Ala Asp Val Asp Gly Tyr Arg Ar #g Phe His Asp Tyr Ala        115           #       120           #       125Glu Glu Val Tyr Arg Glu Gly Tyr Leu Lys Le #u Gly Thr Thr Pro Phe    130               #   135               #   140Leu Lys Leu Gly Gln Met Leu Asn Ala Ala Pr #o Ala Leu Met Arg Leu145                 1 #50                 1 #55                 1 #60Gln Ala Tyr Arg Ser Val His Ser Met Val Al #a Arg Phe Ile Gln Asp                165   #               170   #               175Pro His Leu Arg Gln Ala Phe Ser Phe His Th #r Leu Leu Val Gly Gly            180       #           185       #           190Asn Pro Phe Ser Thr Ser Ser Ile Tyr Ala Le #u Ile His Ala Leu Glu        195           #       200           #       205Arg Arg Gly Gly Val Trp Phe Ala Lys Gly Gl #y Thr Asn Gln Leu Val    210               #   215               #   220Ala Gly Met Val Ala Leu Phe Glu Arg Leu Gl #y Gly Thr Leu Leu Leu225                 2 #30                 2 #35                 2 #40Asn Ala Arg Val Thr Arg Ile Asp Thr Glu Gl #y Asp Arg Ala Thr Gly                245   #               250   #               255Val Thr Leu Leu Asp Gly Arg Gln Leu Arg Al #a Asp Thr Val Ala Ser            260       #           265       #           270Asn Gly Asp Val Met His Ser Tyr Arg Asp Le #u Leu Gly His Thr Arg        275           #       280           #       285Arg Gly Arg Thr Lys Ala Ala Ile Leu Asn Ar #g Gln Arg Trp Ser Met    290               #   295               #   300Ser Leu Phe Val Leu His Phe Gly Leu Ser Ly #s Arg Pro Glu Asn Leu305                 3 #10                 3 #15                 3 #20Ala His His Ser Val Ile Phe Gly Pro Arg Ty #r Lys Gly Leu Val Asn                325   #               330   #               335Glu Ile Phe Asn Gly Pro Arg Leu Pro Asp As #p Phe Ser Met Tyr Leu            340       #           345       #           350His Ser Pro Cys Val Thr Asp Pro Ser Leu Al #a Pro Glu Gly Met Ser        355           #       360           #       365Thr His Tyr Val Leu Ala Pro Val Pro His Le #u Gly Arg Ala Asp Val    370               #   375               #   380Asp Trp Glu Ala Glu Ala Pro Gly Tyr Ala Gl #u Arg Ile Phe Glu Glu385                 3 #90                 3 #95                 4 #00Leu Glu Arg Arg Ala Ile Pro Asp Leu Arg Ly #s His Leu Thr Val Ser                405   #               410   #               415Arg Ile Phe Ser Pro Ala Asp Phe Ser Thr Gl #u Leu Ser Ala His His            420       #           425       #           430Gly Ser Ala Phe Ser Val Glu Pro Ile Leu Th #r Gln Ser Ala Trp Phe        435           #       440           #       445Arg Pro His Asn Arg Asp Arg Ala Ile Pro As #n Phe Tyr Ile Val Gly    450               #   455               #   460Ala Gly Thr His Pro Gly Ala Gly Ile Pro Gl #y Val Val Gly Ser Ala465                 4 #70                 4 #75                 4 #80Lys Ala Thr Ala Gln Val Met Leu Ser Asp Le #u Ala Val Ala                485   #               490 <210> SEQ ID NO 5<211> LENGTH: 382 <212> TYPE: PRT<213> ORGANISM: Flavobacterium sp. R1534 <400> SEQUENCE: 5Met Ser His Asp Leu Leu Ile Ala Gly Ala Gl #y Leu Ser Gly Ala Leu  1               5  #                 10  #                 15Ile Ala Leu Ala Val Arg Asp Arg Arg Pro As #p Ala Arg Ile Val Met             20      #             25      #             30Leu Asp Ala Arg Ser Gly Pro Ser Asp Gln Hi #s Thr Trp Ser Cys His         35          #         40          #         45Asp Thr Asp Leu Ser Pro Glu Trp Leu Ala Ar #g Leu Ser Pro Ile Arg     50              #     55              #     60Arg Gly Glu Trp Thr Asp Gln Glu Val Ala Ph #e Pro Asp His Ser Arg 65                  # 70                  # 75                  # 80Arg Leu Thr Thr Gly Tyr Gly Ser Ile Glu Al #a Gly Ala Leu Ile Gly                 85  #                 90  #                 95Leu Leu Gln Gly Val Asp Leu Arg Trp Asn Th #r His Val Ala Thr Leu            100       #           105       #           110Asp Asp Thr Gly Ala Thr Leu Thr Asp Gly Se #r Arg Ile Glu Ala Ala        115           #       120           #       125Cys Val Ile Asp Ala Arg Gly Ala Val Glu Th #r Pro His Leu Thr Val    130               #   135               #   140Gly Phe Gln Lys Phe Val Gly Val Glu Ile Gl #u Thr Asp Ala Pro His145                 1 #50                 1 #55                 1 #60Gly Val Glu Arg Pro Met Ile Met Asp Ala Th #r Val Pro Gln Met Asp                165   #               170   #               175Gly Tyr Arg Phe Ile Tyr Leu Leu Pro Phe Se #r Pro Thr Arg Ile Leu            180       #           185       #           190Ile Glu Asp Thr Arg Tyr Ser Asp Gly Gly As #p Leu Asp Asp Gly Ala        195           #       200           #       205Leu Ala Gln Ala Ser Leu Asp Tyr Ala Ala Ar #g Arg Gly Trp Thr Gly    210               #   215               #   220Gln Glu Met Arg Arg Glu Arg Gly Ile Leu Pr #o Ile Ala Leu Ala His225                 2 #30                 2 #35                 2 #40Asp Ala Ile Gly Phe Trp Arg Asp His Ala Gl #n Gly Ala Val Pro Val                245   #               250   #               255Gly Leu Gly Ala Gly Leu Phe His Pro Val Th #r Gly Tyr Ser Leu Pro            260       #           265       #           270Tyr Ala Ala Gln Val Ala Asp Ala Ile Ala Al #a Arg Asp Leu Thr Thr        275           #       280           #       285Ala Ser Ala Arg Arg Ala Val Arg Gly Trp Al #a Ile Asp Arg Ala Asp    290               #   295               #   300Arg Asp Arg Phe Leu Arg Leu Leu Asn Arg Me #t Leu Phe Arg Gly Cys305                 3 #10                 3 #15                 3 #20Pro Pro Asp Arg Arg Tyr Arg Leu Leu Gln Ar #g Phe Tyr Arg Leu Pro                325   #               330   #               335Gln Pro Leu Ile Glu Arg Phe Tyr Ala Gly Ar #g Leu Thr Leu Ala Asp            340       #           345       #           350Arg Leu Arg Ile Val Thr Gly Arg Pro Pro Il #e Pro Leu Ser Gln Ala        355           #       360           #       365Val Arg Cys Leu Pro Glu Arg Pro Leu Leu Gl #n Glu Arg Ala    370               #   375               #   380 <210> SEQ ID NO 6<211> LENGTH: 169 <212> TYPE: PRT<213> ORGANISM: Flavobacterium sp. R1534 <400> SEQUENCE: 6Met Ser Thr Trp Ala Ala Ile Leu Thr Val Il #e Leu Thr Val Ala Ala  1               5  #                 10  #                 15Met Glu Leu Thr Ala Tyr Ser Val His Arg Tr #p Ile Met His Gly Pro             20      #             25      #             30Leu Gly Trp Gly Trp His Lys Ser His His As #p Glu Asp His Asp His         35          #         40          #         45Ala Leu Glu Lys Asn Asp Leu Tyr Gly Val Il #e Phe Ala Val Ile Ser     50              #     55              #     60Ile Val Leu Phe Ala Ile Gly Ala Met Gly Se #r Asp Leu Ala Trp Trp 65                  # 70                  # 75                  # 80Leu Ala Val Gly Val Thr Cys Tyr Gly Leu Il #e Tyr Tyr Phe Leu His                 85  #                 90  #                 95Asp Gly Leu Val His Gly Arg Trp Pro Phe Ar #g Tyr Val Pro Lys Arg            100       #           105       #           110Gly Tyr Leu Arg Arg Val Tyr Gln Ala His Ar #g Met His His Ala Val        115           #       120           #       125His Gly Arg Glu Asn Cys Val Ser Phe Gly Ph #e Ile Trp Ala Pro Ser    130               #   135               #   140Val Asp Ser Leu Lys Ala Glu Leu Lys Arg Se #r Gly Ala Leu Leu Lys145                 1 #50                 1 #55                 1 #60Asp Arg Glu Gly Ala Asp Arg Asn Thr                 165<210> SEQ ID NO 7 <211> LENGTH: 52 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial #Sequence: Primer #100 <400> SEQUENCE: 7tatatactag taagaggaga aattacatat gacgcccaag cagcagcaat tc#             52 <210> SEQ ID NO 8 <211> LENGTH: 32 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial #Sequence: Primer #101 <400> SEQUENCE: 8tatatacccg ggtcagccgc gacggcctgt gg        #                  #          32 <210> SEQ ID NO 9 <211> LENGTH: 50 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial #Sequence: Primer #104 <400> SEQUENCE: 9tatatgaatt caagaggaga aattacatat gagcacttgg gccgcaatcc  #              50 <210> SEQ ID NO 10 <211> LENGTH: 21 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial #Sequence: Primer #105 <400> SEQUENCE: 10gtttcagctc tgccttgagg c            #                  #                   #21 <210> SEQ ID NO 11 <211> LENGTH: 62<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial #Sequence: Primer MUT1 <400> SEQUENCE: 11gcgaaggggc ggatcgcaat acgtgaaagg aggacacgtg atgagccatg at#ctgctgat     60 cg                   #                  #                   #              62 <210> SEQ ID NO 12<211> LENGTH: 63 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of Artificial #Sequence: Primer MUT2 <400> SEQUENCE: 12gccccctgct gcaggagaga gcttgaaagg aggcaattga gatgagttcc gc#catcgtca     60 tcg                   #                  #                   #             63 <210> SEQ ID NO 13 <211> LENGTH: 70<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial #Sequence: Primer MUT3 <400> SEQUENCE: 13ggtcatgctg tcggacctgg ccgtcgcttg aaaggaggat ccaatcatga cc#gatctgac     60 ggcgacttcc                 #                  #                   #        70 <210> SEQ ID NO 14 <211> LENGTH: 44<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial #Sequence: Primer MUT5 <400> SEQUENCE: 14atatatctca attgcctcct ttcaagctct ctcctgcagc aggg    #                  # 44 <210> SEQ ID NO 15 <211> LENGTH: 42 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial #Sequence: Primer MUT6 <400> SEQUENCE: 15atgattggat cctcctttca agcgacggcc aggtccgaca gc     #                  #  42 <210> SEQ ID NO 16 <211> LENGTH: 22 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: Primer      CAR17 <400> SEQUENCE: 16 cagaacccat cacctgcccg tc           #                   #                 22 <210> SEQ ID NO 17<211> LENGTH: 27 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of Artificial #Sequence: Primer CATe <400> SEQUENCE: 17cgcgaattct cgccggcaat agttacc           #                  #             27 <210> SEQ ID NO 18 <211> LENGTH: 60 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial #Sequence: Primer CAT4 <400> SEQUENCE: 18gtcacatgca tgcatgttac gagctcataa gcatgtgacg tcttcaacta ac#ggggcagg     60 <210> SEQ ID NO 19 <211> LENGTH: 34 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial #Sequence: Primer CS1 <400> SEQUENCE: 19agcttggatc cttaagtact ctagagttta aacg        #                  #        34 <210> SEQ ID NO 20 <211> LENGTH: 34 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: Primer      CS2 <400> SEQUENCE: 20aattcgttta aactctagag tacttaagga tcca        #                  #        34 <210> SEQ ID NO 21 <211> LENGTH: 46 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: Primer      MUT7 <400> SEQUENCE: 21tcgaccctag gcacgtgacg cgtcaattgg atccgcatgc aagctt   #                 46 <210> SEQ ID NO 22 <211> LENGTH: 46 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: Primer      MUT8 <400> SEQUENCE: 22gatcaagctt gcatgcggat ccaattgacg cgtcacgtgc ctaggg   #                 46 <210> SEQ ID NO 23 <211> LENGTH: 84 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: Primer      MUT9 <400> SEQUENCE: 23gtgtcctcct ttcacgtatt gcgatccgcc ccttcgcggt ccttcagcag cg#cgcccgag     60 cgtttcagct ctgccttgag gctg          #                   #                84 <210> SEQ ID NO 24<211> LENGTH: 88 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of Artificial #Sequence: Primer       MUT10 <400> SEQUENCE: 24tcgacagcct caaggcagag ctgaaacgct cgggcgcgct gctgaaggac cg#cgaagggg     60 cggatcgcaa tacgtgaaag gaggacac         #                   #             88 <210> SEQ ID NO 25 <211> LENGTH: 17<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: Primer      MUT11 <400> SEQUENCE: 25 taagaaaccc tccttta             #                   #                   #   17 <210> SEQ ID NO 26<211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of Artificial #Sequence: Primer       MUT12 <400> SEQUENCE: 26ctagtaaagg agggtttct              #                  #                   # 19 <210> SEQ ID NO 27 <211> LENGTH: 11233<212> TYPE: DNA <213> ORGANISM: Unknown <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or#ganism: Plasmid pZea4 <400> SEQUENCE: 27ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aa#atcagctc     60attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aa#tagaccga    120gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga ac#gtggact     180caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aa#ccatcac     240ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ct#aaagggag    300cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aa#gggaagaa    360agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gc#gtaaccac    420cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tc#aggctgcg    480caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tg#gcgaaagg    540gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt ca#cgacgttg    600taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tg#gagctcca    660ccgcggtggc ggccgctcta gtggatccgc gcctggccgt tcgcgatcag ca#gccgccct    720tgcggatcgg tcagcatcat ccccatgaac cgcagcgcac gacgcagcgc gc#gccccaga    780tcgggcgcgt ccagcacggc atgcgccatc atcgcgaagg cccccggcgg ca#tggggcgc    840gtgcccattc cgaagaactc gcagcctgtc cgctgcgcaa ggtcgcgcca ga#tcgcgccg    900tattccgatg cagtgacggg cccgatgcgc gtgggcccgc cctgccccgc cg#ccaccagc    960gcatcgcgca cgaacccttc cgagatgatg tgctgatcca tggcccgtca tt#gcaaaacc   1020gatcaccgat cctgtcgcgt gatggcattg tttgcaatgc cccgagggct ag#gatggcgc   1080gaaggatcaa gggggggaga gacatggaaa tcgagggacg ggtctttgtc gt#cacgggcg   1140ccgcatcggg tctgggggcg gcctcggcgc ggatgctggc ccaaggcggc gc#gaaggtcg   1200tgctggccga tctggcggaa ccgaaggacg cgcccgaagg cgcggttcac gc#ggcctgcg   1260acgtgaccga cgcgaccgct gcgcagacgg ccatcgcgct ggcgaccgac cg#cttcggca   1320ggctggacgg ccttgtgaac tgcgcgggca tcgcgccggc cgaacggatg ct#gggccgcg   1380acgggccgca tggactggac agctttgccc gtgcggtcac gatcaacctg at#cggcagct   1440tcaacatggc ccgccttgca gccgaggcga tggcccggaa cgagcccgtc cg#gggcgagc   1500gtggcgtgat cgtcaacacg gcctcgatcg cggcgcagga cggacagatc gg#acaggtcg   1560cctatgcggc cagcaaggcg ggcgtggcgg gcatgacgct gccgatggcc cg#cgaccttg   1620cgcggcacgg catccgcgtc atgaccatcg cgcccggcat cttccgcacc cc#gatgctgg   1680aggggctgcc gcaggacgtt caggacagcc tgggcgcggc ggtgcccttc cc#ctcgcggc   1740tgggagagcc gtcggaatac gcggcgctgt tgcaccacat catcgcgaac cc#catgctga   1800acggagaggt catccgcctc gacggcgcat tgcgcatggc ccccaagtga ag#gagcgttt   1860catggacccc atcgtcatca ccggcgcgat gcgcaccccg atgggggcat tc#cagggcga   1920tcttgccgcg atggatgccc cgacccttgg cgcggacgcg atccgcgccg cg#ctgaacgg   1980cctgtcgccc gacatggtgg acgaggtgct gatgggctgc gtcctcgccg cg#ggccaggg   2040tcaggcaccg gcacgtcagg cggcgcttgg cgccggactg ccgctgtcga cg#ggcacgac   2100caccatcaac gagatgtgcg gatcgggcat gaaggccgcg atgctgggcc at#gacctgat   2160cgccgcggga tcggcgggca tcgtcgtcgc cggcgggatg gagagcatgt cg#aacgcccc   2220ctacctgctg cccaaggcgc ggtcggggat gcgcatgggc catgaccgtg tg#ctggatca   2280catgttcctc gacgggttgg aggacgccta tgacaagggc cgcctgatgg gc#accttcgc   2340cgaggattgc gccggcgatc acggtttcac ccgcgaggcg caggacgact at#gcgctgac   2400cagcctggcc cgcgcgcagg acgccatcgc cagcggtgcc ttcgccgccg ag#atcgcgcc   2460cgtgaccgtc acggcacgca aggtgcagac caccgtcgat accgacgaga tg#cccggcaa   2520ggcccgcccc gagaagatcc cccatctgaa gcccgccttc cgtgacggtg gc#acggtcac   2580ggcggcgaac agctcgtcga tctcggacgg ggcggcggcg ctggtgatga tg#cgccagtc   2640gcaggccgag aagctgggcc tgacgccgat cgcgcggatc atcggtcatg cg#acccatgc   2700cgaccgtccc ggcctgttcc cgacggcccc catcggcgcg atgcgcaagc tg#ctggaccg   2760cacggacacc cgccttggcg attacgacct gttcgaggtg aacgaggcat tc#gccgtcgt   2820cgccatgatc gcgatgaagg agcttggcct gccacacgat gccacgaaca tc#aacggcgg   2880ggcctgcgcg cttgggcatc ccatcggcgc gtcgggggcg cggatcatgg tc#acgctgct   2940gaacgcgatg gcggcgcggg gcgcgacgcg cggggccgca tccgtctgca tc#ggcggggg   3000cgaggcgacg gccatcgcgc tggaacggct gagctaattc atttgcgcga at#ccgcgttt   3060ttcgtgcacg atgggggaac cggaaacggc cacgcctgtt gtggttgcgt cg#acctgtct   3120tcgggccatg cccgtgacgc gatgtggcag gcgcatgggg cgttgccgat cc#ggtcgcat   3180gactgacgca acgaaggcac cgatgacgcc caagcagcaa ttccccctac gc#gatctggt   3240cgagatcagg ctggcgcaga tctcgggcca gttcggcgtg gtctcggccc cg#ctcggcgc   3300ggccatgagc gatgccgccc tgtcccccgg caaacgcttt cgcgccgtgc tg#atgctgat   3360ggtcgccgaa agctcgggcg gggtctgcga tgcgatggtc gatgccgcct gc#gcggtcga   3420gatggtccat gccgcatcgc tgatcttcga cgacatgccc tgcatggacg at#gccaggac   3480ccgtcgcggt cagcccgcca cccatgtcgc ccatggcgag gggcgcgcgg tg#cttgcggg   3540catcgccctg atcaccgagg ccatgcggat tttgggcgag gcgcgcggcg cg#acgccgga   3600tcagcgcgca aggctggtcg catccatgtc gcgcgcgatg ggaccggtgg gg#ctgtgcgc   3660agggcaggat ctggacctgc acgcccccaa ggacgccgcc gggatcgaac gt#gaacagga   3720cctcaagacc ggcgtgctgt tcgtcgcggg cctcgagatg ctgtccatta tt#aagggtct   3780ggacaaggcc gagaccgagc agctcatggc cttcgggcgt cagcttggtc gg#gtcttcca   3840gtcctatgac gacctgctgg acgtgatcgg cgacaaggcc agcaccggca ag#gatacggc   3900gcgcgacacc gccgcccccg gcccaaaggg cggcctgatg gcggtcggac ag#atgggcga   3960cgtggcgcag cattaccgcg ccagccgcgc gcaactggac gagctgatgc gc#acccggct   4020gttccgcggg gggcagatcg cggacctgct ggcccgcgtg ctgccgcatg ac#atccgccg   4080cagcgcctag gcgcgcggtc gggtccacag gccgtcgcgg ctgatttcgc cg#ccgcgcag   4140gcgcgatgcg gccgcgtcca agcctccgcg cgccagaagc ccgatcttgg ca#gccttcga   4200cgtgctgatc cgctggcgat aggcctcggg gccaccctgc cggatgcgcg tc#ccgattgc   4260gcgatagata cgcagcgcgg cggcgatcga ccacgcgcag cgcggcggca ga#tgcggaag   4320cccctgccgc gccgaggcat aatagggctc ggccgcgtca agcaggcgga tg#atgacgga   4380atagagcgcg tccgaaggca ccggaccctc aaccgtcgcc cccgcctcgg cc#agccagtc   4440ggcaggcaga tagcagcgcc cgatggcggc atcgtcgatc acgtcgcgag cg#atgttcgt   4500cagctggaac gcaaggccca gatcgcaggc gcgatccagc accgcatcgt cc#tgcacgcc   4560catcacccgc gccatcatca cgcccacgac ccccgcgacg tggtaggaat at#tccagcac   4620gtcatccagg ctgcggtatt cgcgatccgc gacatccatc gcgaaaccct cg#atcaggtc   4680catcggccaa aggtccggga aatcatgccg ccgggcgacc tggcgcagcg cc#gcgaaggg   4740cggcgacatc gggccgtcct cgtgcagcgc ggccagcgtg tcggcgcgca gc#gcccccag   4800ccgcgcctgt gggtcgccgc ccgcctcggg ggcagaaccc atcacctgcc cg#tcgatcac   4860gtcatccgca tgcctgcacc aggcatagag catgaccgta tcctcgcgga tg#ccgggcgg   4920catcagcttg gccgcctgcg cgaagctttg cgaaccctgc gcgatggccg ct#tcggaagt   4980cgccgtcaga tcggtcatgc gacggccagg tccgacagca tgacctgcgc cg#tggccttg   5040gcgctgccaa cgacacccgg gatgcccgca cccggatgcg tgcccgcccc ca#cgatgtag   5100aagttcggga tcgcgcggtc gcggttatgc gggcggaacc aggcggattg cg#tcaggatc   5160ggctcgaccg agaaggcgct gccgtgatgg gccgacagtt cggtgctgaa at#cggcgggg   5220ctgaagatgc ggctgacggt caggtgcttg cgcaggtcgg ggatggcgcg gc#gctccagt   5280tcctcgaaga tgcgctcggc atagcccggg gcctcggctt cccaatcgac at#cggcgcgg   5340cccagatgcg gaacgggcgc aaggacgtaa tgcgtggaca tcccctcggg gg#ccaggctg   5400ggatcggtca cgcagggcga atgcagatac atcgagaaat cgtccggcag gc#gtggcccg   5460ttgaagatct cgttcaccag ccccttgtag cgcgggccga agatgacgct gt#ggtgggcc   5520aggttctcgg ggcgcttgga caggccgaaa tgcagcacga acagcgacat cg#accagcgc   5580tgccggttca ggatcgcggc cttggtgcgc ccgcggcggg tatggcccag ca#ggtcgcga   5640tagctgtgca tcacgtcgcc gttgctggcc accgtatccg cgcgcaactg cc#gcccgtcc   5700agcagcgtga cgcccgtggc gcgatcgccc tcggtgtcga tccgcgtgac gc#gggcattc   5760agcagcagcg tgccgccaag acgctcgaac agggcgacca tgcccgcgac ca#gctggttg   5820gtgccgccct tggcgaacca gacgccgccg cgccgttcca gcgcatggat ca#gcgcatag   5880atcgagctgg tcgaaaacgg gttcccgccg accagcagcg tgtggaacga ga#aggcctgc   5940cgcagatgcg ggtcctggat gaagcgcgcc accatgctgt ggaccgagcg gt#atgcctgc   6000aggcgcatca gcgccggcgc ggcgttcagc atctggccca gcttcaggaa gg#gcgtggtc   6060cccagcttca gatacccctc gcgatagacc tcctcggcgt aatcgtggaa gc#ggcgatag   6120ccatcgacat cggcgggatt gaaggaggcg acctggcgga tcagctcgtc gt#cgtcgttc   6180acgtattcga agctgcggcc gtccgcccat gtcagccggt agaagggcga ga#ccggcagc   6240agcgtcacgt cacgctccat cggttggccg ctgagggccc acagctctcg ca#ggctgtcg   6300gggtcggtca cgaccgtcgg gcctgcatcg aagacgtggc cctgatcgtt cc#agacatag   6360gcgcggccgc cgggcttgtc gcgggcctcg acgatggtgg tcgcgatgcc gg#ccgattgc   6420aggcggatgg caagcgcaag cccgccgaaa cctgcgccga tgacgatggc gg#aactcatg   6480ctctctcctg cagcaggggg cgttcgggca ggcagcgcac ggcctgcgac ag#cggaatgg   6540gcgggcgtcc ggtgacgatg cgaagccggt cggccaatgt caggcgcccg gc#atagaagc   6600gctcgatcag cggctgcggc aggcggtaga accgctgcag caggcgatag cg#acggtcgg   6660gcgggcagcc gcggaacagc atccggttca gcagccgcag gaagcggtcg cg#atccgcgc   6720gatcgatggc ccagccgcgc accgcgcgac gggcggacgc ggtcgtcagg tc#gcgcgccg   6780cgatggcatc cgcgacctgc gcggcatagg gcagcgaata tccggtgacg gg#gtggaaca   6840gccctgcccc cagcccaacc ggcaccgccc cctgcgcgtg gtcgcgccag aa#gcctatgg   6900cgtcatgggc cagcgcgatg ggcaggatgc ccctttcgcg ccgcatctcc tg#cccggtcc   6960agccccgcct ggcggcatag tccagcgacg cctgcgccag cgcgccatcg tc#cagatcgc   7020cgccgtcgct gtagcgcgta tcctcgatca ggatgcgggt gggactgaag gg#cagcagat   7080agatgaagcg gtacccgtcc atctgcggaa cggtcgcgtc catgatcatc gg#gcgctcga   7140cgccatgggg ggcgtcggtc tcgatctcga cgcccacgaa tttctggaaa cc#cacggtca   7200ggtgcggggt ctcgacggca ccacgggcgt cgatcacgca ggcagcctcg at#ccgcgagc   7260cgtccgtcag cgtcgcgccg gtatcgtcca gcgtcgcgac atgcgtattc ca#ccgcagat   7320cgacaccctg cagcagcccg atcagcgcgc ccgcctcgat cgagccatag cc#tgtcgtca   7380ggcggcgcga atggtcggga aacgcgacct cctgatccgt ccattcgccg cg#acgaatgg   7440gcgacaggcg cgccagccat tcgggcgaaa gatccgtgtc gtggcaggac ca#ggtgtgct   7500ggtccgaggg gccggaccgc gcgtcgagca tcacgatgcg cgcatccggt ct#gcggtcgc   7560gaacggcaag cgcgatcagc gcaccggaca gccccgcgcc cgcgatcagc ag#atcatggc   7620tcatgtattg cgatccgccc cttcgcggtc cttcagcagc gcgcccgagc gt#ttcagctc   7680tgccttgagg ctgtcgaccg agggcgccca gatgaaaccg aagctgacgc ag#ttctcgcg   7740gccatggacc gcgtgatgca tcctgtgtgc ctggtagacg cgacgaagat ag#ccgcgctt   7800ggggacatag cggaacggcc agcgcccatg caccaagccg tcatgcagga aa#tagtagat   7860cagcccgtag caggtgaccc ccaccgccag ccaccaggcc agatccgacc cc#atcgcgcc   7920gatcgcgaac agcacgatcg agattaccgc gaagatgacg ccatagaggt cg#ttcttctc   7980gagcgcgtgg tcgtgatcct cgtcgtggtg cgatttatgc cagccccagc cc#agggggcc   8040atgcatgatc caccgatgga cggagtaggc cgtcagctcc atcgcggcga cg#gtcaggat   8100gacggtcagg attgcggccc aagtgctcat gccggcccct tgcttgatat ga#cagggaac   8160aggctacgct gccgcgcggt gcatgaccag cccatcgggg tgcgaccaaa gg#gcatcgcg   8220tgacatctgc gttcagggct cataggcgga tcatccgtga cattcgccgc cg#aacgcggc   8280aggcgcatca cgcgttccgt cgctggaaat attaatgttt tcccgaagat gg#tcggggcg   8340agaggattcg aacctccgac ctacggtacc caaaaccgtc gcgctaccag gc#tgcgctac   8400gccccgactg cggaaggctt tagccgattg ttccggcaag ggaaagacct ag#tcgcaggc   8460caggaccgca ttgtcgccca tgcccggatg cgccatcggc tgaccgggct tc#aggccaag   8520gcgatccgcc tctccgcccg cgatttcgag gacgaacagc cggtcggggt cc#ggatcgcc   8580gaccgccgcg cccggaatgg gcgtctcgtc cagcgggcgc gcattgcggt gg#atgtggcg   8640gatgacgccg gtttcatccg caaagaccat gtccagcggg atcagtgtgt tg#cgcatcca   8700gaaggacacc ggctggggcg attcgtagat gaacagcatt ccggtgcccg ca#ggcagctc   8760cttgcggaac atcaggccct gcgcgcgctc ttcggggctg tccgcgacct cg#acccgaaa   8820cccgagcgtt tccgcaccgg tatcgacgac aagactgccg ggcgcgcatt cc#accgccgc   8880cgcggcggcg ggcatcagga ccgcaagaag cgctgcggcc ttactcggcc ac#atgggcaa   8940gataggactg ctcggcgccg agatcccccg ggctgcagga attcgatatc aa#gcttatcg   9000ataccgtcga cctcgagggg gggcccggta cccagctttt gttcccttta gt#gagggtta   9060attgcgcgct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg tt#atccgctc   9120acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tg#cctaatga   9180gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gg#gaaacctg   9240tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gc#gtattggg   9300cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gc#ggcgagcg   9360gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga ta#acgcagga   9420aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cg#cgttgctg   9480gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ct#caagtcag   9540aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aa#gctccctc   9600gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tc#tcccttcg   9660ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gt#aggtcgtt   9720cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cg#ccttatcc   9780ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact gg#cagcagcc   9840actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt ct#tgaagtgg   9900tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct gc#tgaagcca   9960gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cg#ctggtagc  10020ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tc#aagaagat  10080cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg tt#aagggatt  10140ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aa#aatgaagt  10200tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca at#gcttaatc  10260agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ct#gactcccc  10320gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tg#caatgata  10380ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc ag#ccggaagg  10440gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat ta#attgttgc  10500cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tg#ccattgct  10560acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cg#gttcccaa  10620cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ct#ccttcggt  10680cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt ta#tggcagca  10740ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tg#gtgagtac  10800tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cc#cggcgtca  10860atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tg#gaaaacgt  10920tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc ga#tgtaaccc  10980actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tg#ggtgagca  11040aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa at#gttgaata  11100ctcatactct tcctttttca atattattga agcatttatc agggttattg tc#tcatgagc  11160ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg ca#catttccc  11220 cgaaaagtgc cac               #                  #                   #  11233 <210> SEQ ID NO 28 <211> LENGTH: 726<212> TYPE: DNA <213> ORGANISM: Alcaligenes PC-1 <400> SEQUENCE: 28atgtccggtc gtaaaccggg taccaccggt gacaccatcg ttaacctggg tc#tgaccgct     60gctatcctgc tgtgctggct ggttctgcac gctttcaccc tgtggctgct gg#acgctgct    120gctcacccgc tgctggctgt tctgtgcctg gctggtctga cctggctgtc cg#ttggtctg    180ttcatcatcg ctcacgacgc tatgcacggt tccgttgttc cgggtcgtcc gc#gggctaac    240gctgctatcg gtcagctggc tctgtggctg tacgctggtt tctcctggcc ga#aactgatc    300gctaaacaca tgacccacca ccgtcacgct ggtaccgaca acgacccgga ct#tcggtcac    360ggtggtccgg ttcgttggta cggttccttc gtttccacct acttcggttg gc#gtgaaggt    420ctgctgctgc cggttatcgt taccacctac gctctgatcc tgggtgaccg tt#ggatgtac    480gttatcttct ggccggttcc ggctgttctg gcttccatcc agatcttcgt tt#tcggtacc    540tggctgccgc accgtccggg tcacgacgac ttcccggacc gtcacaacgc tc#gttccacc    600ggtatcggtg acccgctgtc cctgctgacc tgcttccact tcggtggtta cc#accacgaa    660caccacctgc acccgcacgt tccgtggtgg cgtctgccgc gtacccgtaa aa#ccggtggt    720 cgtgct                  #                  #                   #          726 <210> SEQ ID NO 29 <211> LENGTH: 242<212> TYPE: PRT <213> ORGANISM: Alcaligenes PC-1 <400> SEQUENCE: 29Met Ser Gly Arg Lys Pro Gly Thr Thr Gly As #p Thr Ile Val Asn Leu  1               5  #                 10  #                 15Gly Leu Thr Ala Ala Ile Leu Leu Cys Trp Le #u Val Leu His Ala Phe             20      #             25      #             30Thr Leu Trp Leu Leu Asp Ala Ala Ala His Pr #o Leu Leu Ala Val Leu         35          #         40          #         45Cys Leu Ala Gly Leu Thr Trp Leu Ser Val Gl #y Leu Phe Ile Ile Ala     50              #     55              #     60His Asp Ala Met His Gly Ser Val Val Pro Gl #y Arg Pro Arg Ala Asn 65                  # 70                  # 75                  # 80Ala Ala Ile Gly Gln Leu Ala Leu Trp Leu Ty #r Ala Gly Phe Ser Trp                 85  #                 90  #                 95Pro Lys Leu Ile Ala Lys His Met Thr His Hi #s Arg His Ala Gly Thr            100       #           105       #           110Asp Asn Asp Pro Asp Phe Gly His Gly Gly Pr #o Val Arg Trp Tyr Gly        115           #       120           #       125Ser Phe Val Ser Thr Tyr Phe Gly Trp Arg Gl #u Gly Leu Leu Leu Pro    130               #   135               #   140Val Ile Val Thr Thr Tyr Ala Leu Ile Leu Gl #y Asp Arg Trp Met Tyr145                 1 #50                 1 #55                 1 #60Val Ile Phe Trp Pro Val Pro Ala Val Leu Al #a Ser Ile Gln Ile Phe                165   #               170   #               175Val Phe Gly Thr Trp Leu Pro His Arg Pro Gl #y His Asp Asp Phe Pro            180       #           185       #           190Asp Arg His Asn Ala Arg Ser Thr Gly Ile Gl #y Asp Pro Leu Ser Leu        195           #       200           #       205Leu Thr Cys Phe His Phe Gly Gly Tyr His Hi #s Glu His His Leu His    210               #   215               #   220Pro His Val Pro Trp Trp Arg Leu Pro Arg Th #r Arg Lys Thr Gly Gly225                 2 #30                 2 #35                 2 #40Arg Ala <210> SEQ ID NO 30 <211> LENGTH: 1261 <212> TYPE: DNA<213> ORGANISM: Alcaligenes PC-1 <400> SEQUENCE: 30actgtagtct gcgcggatcg ccggtccggg ggacaagata tgagcgcaca tg#ccctgccc     60aaggcagatc tgaccgccac cagtttgatc gtctcgggcg gcatcatcgc cg#cgtggctg    120gccctgcatg tgcatgcgct gtggtttctg gacgcggcgg cgcatcccat cc#tggcggtc    180gcgaatttcc tggggctgac ctggctgtcg gtcggtctgt tcatcatcgc gc#atgacgcg    240atgcatgggt cggtcgtgcc ggggcgcccg cgcgccaatg cggcgatggg cc#agcttgtc    300ctgtggctgt atgccggatt ttcctggcgc aagatgatcg tcaagcacat gg#cccatcat    360cgccatgccg gaaccgacga cgacccagat ttcgaccatg gcggcccggt cc#gctggtac    420gcccgcttca tcggcaccta tttcggctgg cgcgaggggc tgctgctgcc cg#tcatcgtg    480acggtctatg cgctgatgtt gggggatcgc tggatgtacg tggtcttctg gc#cgttgccg    540tcgatcctgg cgtcgatcca gctgttcgtg ttcggcatct ggctgccgca cc#gccccggc    600cacgacgcgt tcccggaccg ccacaatgcg cggtcgtcgc ggatcagcga cc#ccgtgtcg    660ctgctgacct gctttcactt tggcggttat catcacgaac accacctgca cc#cgacggtg    720ccttggtggc gcctgcccag cacccgcacc aagggggaca ccgcatgacc aa#tttcctga    780tcgtcgtcgc caccgtgctg gtgatggagc tgacggccta ttccgtccac cg#ctggatca    840tgcacggccc cttgggctgg ggctggcaca agtcccacca cgaggaacac ga#ccacgcgc    900tggaaaagaa cgacctgtac ggcctggtct ttgcggtgat cgccacggtg ct#gttcacgg    960tgggctggat ctgggcaccg gtcctgtggt ggatcgcctt gggcatgacc gt#ctacgggc   1020tgatctattt cgtcctgcat gacgggctgg tgcatcagcg ctggccgttc cg#ctatatcc   1080ctcgcaaggg ctatgccaga cgcctgtatc aggcccaccg cctgcaccac gc#ggtcgagg   1140ggcgcgacca ttgcgtcagc ttcggcttca tctatgcgcc gccggtcgac aa#gctgaagc   1200aggacctgaa gacgtcgggc gtgctgcggg ccgaggcgca ggagcgcacg tg#acccatga   1260 c                   #                  #                   #             1261 <210> SEQ ID NO 31<211> LENGTH: 1458 <212> TYPE: DNA <213> ORGANISM: E-396 <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or #ganism: Unkown<400> SEQUENCE: 31atgagcgcac atgccctgcc caaggcagat ctgaccgcca ccagtttgat cg#tctcgggc     60tactcgcgtg tacgggacgg gttccgtcta gactggcggt ggtcaaacta gc#agagcccg    120ggcatcatcg ccgcgtggct ggccctgcat gtgcatgcgc tgtggtttct gg#acgcggcg   180ccgtagtagc ggcgcaccga ccgggacgta cacgtacgcg acaccaaaga cc#tgcgccgc   240gcgcatccca tcctggcggt cgcgaatttc ctggggctga cctggctgtc gg#tcggtctg   300cgcgtagggt aggaccgcca gcgcttaaag gaccccgact ggaccgacag cc#agccagac   360ttcatcatcg cgcatgacgc gatgcatggg tcggtcgtgc cggggcgccc gc#gcgccaat   420aagtagtagc gcgtactgcg ctacgtaccc agccagcacg gccccgcggg cg#cgcggtta   480gcggcgatgg gccagcttgt cctgtggctg tatgccggat tttcctggcg ca#agatgatc   540cgccgctacc cggtcgaaca ggacaccgac atacggccta aaaggaccgc gt#tctactag   600gtcaagcaca tggcccatca tcgccatgcc ggaaccgacg acgacccaga tt#tcgaccat   660cagttcgtgt accgggtagt agcggtacgg ccttggctgc tgctgggtct aa#agctggta   720ggcggcccgg tccgctggta cgcccgcttc atcggcacct atttcggctg gc#gcgagggg   780ccgccgggcc aggcgaccat gcgggcgaag tagccgtgga taaagccgac cg#cgctcccc   840ctgctgctgc ccgtcatcgt gacggtctat gcgctgatgt tgggggatcg ct#ggatgtac   900gacgacgacg ggcagtagca ctgccagata cgcgactaca accccctagc ga#cctacatg   960gtggtcttct ggccgttgcc gtcgatcctg gcgtcgatcc agctgttcgt gt#tcggcatc   1020caccagaaga ccggcaacgg cagctaggac cgcagctagg tcgacaagca ca#agccgtag   1080tggctgccgc accgccccgg ccacgacgcg ttcccggacc gccacaatgc gc#ggtcgtcg   1140accgacggcg tggcggggcc ggtgctgcgc aagggcctgg cggtgttacg cg#ccagcagc   1200cggatcagcg accccgtgtc gctgctgacc tgctttcact ttggcggtta tc#atcacgaa   1260gcctagtcgc tggggcacag cgacgactgg acgaaagtga aaccgccaat ag#tagtgctt   1320caccacctgc acccgacggt gccttggtgg cgcctgccca gcacccgcac ca#agggggac   1380gtggtggacg tgggctgcca cggaaccacc gcggacgggt cgtgggcgtg gt#tccccctg   1440 accgcatgat ggcgtact              #                  #                   #1458 <210> SEQ ID NO 32 <211> LENGTH: 242<212> TYPE: PRT <213> ORGANISM: E-396 <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or #ganism: Unkown<400> SEQUENCE: 32 Met Ser Ala His Ala Leu Pro Lys Ala Asp Le#u Thr Ala Thr Ser Leu   1               5  #                 10 #                 15 Ile Val Ser Gly Gly Ile Ile Ala Ala Trp Le#u Ala Leu His Val His              20      #             25     #             30 Ala Leu Trp Phe Leu Asp Ala Ala Ala His Pr#o Ile Leu Ala Val Ala          35          #         40         #         45 Asn Phe Leu Gly Leu Thr Trp Leu Ser Val Gl#y Leu Phe Ile Ile Ala      50              #     55             #     60 His Asp Ala Met His Gly Ser Val Val Pro Gl#y Arg Pro Arg Ala Asn  65                  # 70                 # 75                  # 80 Ala Ala Met Gly Gln Leu Val Leu Trp Leu Ty#r Ala Gly Phe Ser Trp                  85  #                 90 #                 95 Arg Lys Met Ile Val Lys His Met Ala His Hi#s Arg His Ala Gly Thr             100       #           105      #           110 Asp Asp Asp Pro Asp Phe Asp His Gly Gly Pr#o Val Arg Trp Tyr Ala         115           #       120          #       125 Arg Phe Ile Gly Thr Tyr Phe Gly Trp Arg Gl#u Gly Leu Leu Leu Pro     130               #   135              #   140 Val Ile Val Thr Val Tyr Ala Leu Met Leu Gl#y Asp Arg Trp Met Tyr 145                 1 #50                 1#55                 1 #60 Val Val Phe Trp Pro Leu Pro Ser Ile Leu Al#a Ser Ile Gln Leu Phe                 165   #               170  #               175 Val Phe Gly Ile Trp Leu Pro His Arg Pro Gl#y His Asp Ala Phe Pro             180       #           185      #           190 Asp Arg His Asn Ala Arg Ser Ser Arg Ile Se#r Asp Pro Val Ser Leu         195           #       200          #       205 Leu Thr Cys Phe His Phe Gly Gly Tyr His Hi#s Glu His His Leu His     210               #   215              #   220 Pro Thr Val Pro Trp Trp Arg Leu Pro Ser Th#r Arg Thr Lys Gly Asp 225                 2 #30                 2#35                 2 #40 Thr Ala <210> SEQ ID NO 33 <211> LENGTH: 972<212> TYPE: DNA <213> ORGANISM: E-396 <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or #ganism: Unknown<400> SEQUENCE: 33atgaccaatt tcctgatcgt cgtcgccacc gtgctggtga tggagctgac gg#cctattcc     60tactggttaa aggactagca gcagcggtgg cacgaccact acctcgactg cc#ggataagg    120gtccaccgct ggatcatgca cggccccttg ggctggggct ggcacaagtc cc#accacgag    180caggtggcga cctagtacgt gccggggaac ccgaccccga ccgtgttcag gg#tggtgctc    240gaacacgacc acgcgctgga aaagaacgac ctgtacggcc tggtctttgc gg#tgatcgcc    300cttgtgctgg tgcgcgacct tttcttgctg gacatgccgg accagaaacg cc#actagcgg    360acggtgctgt tcacggtggg ctggatctgg gcaccggtcc tgtggtggat cg#ccttgggc    420tgccacgaca agtgccaccc gacctagacc cgtggccagg acaccaccta gc#ggaacccg    480atgaccgtct acgggctgat ctatttcgtc ctgcatgacg ggctggtgca tc#agcgctgg    540tactggcaga tgcccgacta gataaagcag gacgtactgc ccgaccacgt ag#tcgcgacc    600ccgttccgct atatccctcg caagggctat gccagacgcc tgtatcaggc cc#accgcctg    660ggcaaggcga tatagggagc gttcccgata cggtctgcgg acatagtccg gg#tggcggac    720caccacgcgg tcgaggggcg cgaccattgc gtcagcttcg gcttcatcta tg#cgccgccg    780gtggtgcgcc agctccccgc gctggtaacg cagtcgaagc cgaagtagat ac#gcggcggc    840gtcgacaagc tgaagcagga cctgaagacg tcgggcgtgc tgcgggccga gg#cgcaggag    900cagctgttcg acttcgtcct ggacttctgc agcccgcacg acgcccggct cc#gcgtcctc    960 cgcacggcgt gc               #                  #                   #      972 <210> SEQ ID NO 34 <211> LENGTH: 162<212> TYPE: PRT <213> ORGANISM: E-396 <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or #ganism: Unknown<400> SEQUENCE: 34 Met Thr Asn Phe Leu Ile Val Val Ala Thr Va#l Leu Val Met Glu Leu   1               5  #                 10 #                 15 Thr Ala Tyr Ser Val His Arg Trp Ile Met Hi#s Gly Pro Leu Gly Trp              20      #             25     #             30 Gly Trp His Lys Ser His His Glu Glu His As#p His Ala Leu Glu Lys          35          #         40         #         45 Asn Asp Leu Tyr Gly Leu Val Phe Ala Val Il#e Ala Thr Val Leu Phe      50              #     55             #     60 Thr Val Gly Trp Ile Trp Ala Pro Val Leu Tr#p Trp Ile Ala Leu Gly  65                  # 70                 # 75                  # 80 Met Thr Val Tyr Gly Leu Ile Tyr Phe Val Le#u His Asp Gly Leu Val                  85  #                 90 #                 95 His Gln Arg Trp Pro Phe Arg Tyr Ile Pro Ar#g Lys Gly Tyr Ala Arg             100       #           105      #           110 Arg Leu Tyr Gln Ala His Arg Leu His His Al#a Val Glu Gly Arg Asp         115           #       120          #       125 His Cys Val Ser Phe Gly Phe Ile Tyr Ala Pr#o Pro Val Asp Lys Leu     130               #   135              #   140 Lys Gln Asp Leu Lys Thr Ser Gly Val Leu Ar#g Ala Glu Ala Gln Glu 145                 1 #50                 1#55                 1 #60 Arg Thr <210> SEQ ID NO 35 <211> LENGTH: 1253<212> TYPE: DNA <213> ORGANISM: E-396 <220> FEATURE:<221> NAME/KEY: unsure <222> LOCATION: (911) <400> SEQUENCE: 35ctgcaggtct gacacggcca gaaggccgcg ccgcgggccg ggggccgccg ca#tcgcgacc     60ggtatccttg ccaagcgccg cctggtcgcc cacaacgtcc agcaggtcgt ca#taggactg    120gaacacccgg cccagctgac ggccaaagtc gatcatctga gtctgctcct cg#gcgtcgaa    180ctccttgatc acggccagca tctccagccc ggcgatgaac agcacgccgg tc#ttcaggtc    240ctgttcctgt tcgacccccg cgccgttctt ggccgcgtgc aggtccaggt cc#tggccggc    300gcacaggccc tgcggcccca gggaccgcga caggatccgc accagctgcg cc#cgcaccgt    360gcccgacgcg ccgcgcgcac cggccagcag ggccatcgcc tcggtgatca gg#gcgatgcc    420gcctagcacg gcgcggcttt cgccatgcgc cacatgggtc gcgggctggc cg#cggcgcag    480cccggcatcg tccatgcagg gcaggtcgtc gaagatcagc gatgcggcat gc#accatctc    540gaccgcgcag gcggcgtcga cgatcgtgtc gcagaccccg cccgaggctt ct#gccgcaag    600cagcatcagc atgccgcgga aacgcttgcc cgacgacagc gcgccatggc tc#atggccgg    660gccgagcggc tgcgacacgg caccgaatcc ctgggcgatc tcctcaagtc tg#gtctgcag    720aagggtggcg tggatcgggt tgacgtctcg tctcatcagt gccttcgcgc tt#gggttctg    780accaggcggg aaggtcaggc cggggcggca ccccgtgacc cgtcatccac cg#tcaacagt    840ccccatgttg gaaggcttca cgcccgattg cgagcctttt cgacggcgac gc#ggggtcgc    900gcggcaattt ntccaacaag gtcagtggac cggcgcgccg atggccgcgc gc#agccaggc    960atccttggcc ggaaacaccc gcgccgcatc atgatcggcc aggatcgtcc gg#cgcgcggc   1020gcggcgcagg tcggccgcgt cacccggatt gtcaagcacc caggccatcg cg#tccgcgac   1080ctcgtccgcg tcgtccatgt cgacgatcag gccgttctcc atgtcgcgga cc#agttcgcg   1140caccggggcg gtgttcgatc gatcaccagg catccggtgg ccatcgcctc gg#acagggac   1200caggaggtga cgaagggctc ggtgaaatag acatgcgcgt gcgaggcctg ca#g          1253 <210> SEQ ID NO 36 <211> LENGTH: 1764 <212> TYPE: DNA<213> ORGANISM: E-396 <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or #ganism: Unknown<400> SEQUENCE: 36atgagacgag acgtcaaccc gatccacgcc acccttctgc agaccagact tg#aggagatc     60tactctgctc tgcagttggg ctaggtgcgg tgggaagacg tctggtctga ac#tcctctag    120gcccagggat tcggtgccgt gtcgcagccg ctcggcccgg ccatgagcca tg#gcgcgctg    180cgggtcccta agccacggca cagcgtcggc gagccgggcc ggtactcggt ac#cgcgcgac    240tcgtcgggca agcgtttccg cggcatgctg atgctgcttg cggcagaagc ct#cgggcggg    300agcagcccgt tcgcaaaggc gccgtacgac tacgacgaac gccgtcttcg ga#gcccgccc    360gtctgcgaca cgatcgtcga cgccgcctgc gcggtcgaga tggtgcatgc cg#catcgctg    420cagacgctgt gctagcagct gcggcggacg cgccagctct accacgtacg gc#gtagcgac    480atcttcgacg acctgccctg catggacgat gccgggctgc gccgcggcca gc#ccgcgacc    540tagaagctgc tggacgggac gtacctgcta cggcccgacg cggcgccggt cg#ggcgctgg    600catgtggcgc atggcgaaag ccgcgccgtg ctaggcggca tcgccctgat ca#ccgaggcg    660gtacaccgcg taccgctttc ggcgcggcac gatccgccgt agcgggacta gt#ggctccgc    720atggccctgc tggccggtgc gcgcggcgcg tcgggcacgg tgcgggcgca gc#tggtgcgg    780taccgggacg accggccacg cgcgccgcgc agcccgtgcc acgcccgcgt cg#accacgcc    840atcctgtcgc ggtccctggg gccgcagggc ctgtgcgccg gccaggacct gg#acctgcac    900taggacagcg ccagggaccc cggcgtcccg gacacgcggc cggtcctgga cc#tggacgtg    960gcggccaaga acggcgcggg ggtcgaacag gaacaggacc tgaagaccgg cg#tgctgttc   1020cgccggttct tgccgcgccc ccagcttgtc cttgtcctgg acttctggcc gc#acgacaag   1080atcgccgggc tggagatgct ggccgtgatc aaggagttcg acgccgagga gc#agactcag   1140tagcggcccg acctctacga ccggcactag ttcctcaagc tgcggctcct cg#tctgagtc   1200atgatcgact ttggccgtca gctgggccgg gtgttccagt cctatgacga cc#tgctggac   1260tactagctga aaccggcagt cgacccggcc cacaaggtca ggatactgct gg#acgacctg   1320gttgtgggcg accaggcggc gcttggcaag gataccggtc gcgatgcggc gg#cccccggc   1380caacacccgc tggtccgccg cgaaccgttc ctatggccag cgctacgccg cc#gggggccg   1440ccgcggcgcg gccttctggc cgtgtcagac ctgcagaacg tgtcccgtca ct#atgaggcc   1500ggcgccgcgc cggaagaccg gcacagtctg gacgtcttgc acagggcagt ga#tactccgg   1560agccgcgccc agctggacgc gatgctgcgc agcaagcgcc ttcaggctcc gg#aaatcgcg   1620tcggcgcggg tcgacctgcg ctacgacgcg tcgttcgcgg aagtccgagg cc#tttagcgc   1680gccctgctgg aacgggttct gccctacgcc gcgcgcgcct agcgggacga cc#ttgcccaa   1740 gacgggatgc ggcgcgcgcg gatc          #                   #              1764 <210> SEQ ID NO 37<211> LENGTH: 293 <212> TYPE: PRT <213> ORGANISM: E-396 <220> FEATURE:<223> OTHER INFORMATION: Description of Unknown Or #ganism: Unkown<400> SEQUENCE: 37 Met Arg Arg Asp Val Asn Pro Ile His Ala Th#r Leu Leu Gln Thr Arg   1               5  #                 10 #                 15 Leu Glu Glu Ile Ala Gln Gly Phe Gly Ala Va#l Ser Gln Pro Leu Gly              20      #             25     #             30 Pro Ala Met Ser His Gly Ala Leu Ser Ser Gl#y Lys Arg Phe Arg Gly          35          #         40         #         45 Met Leu Met Leu Leu Ala Ala Glu Ala Ser Gl#y Gly Val Cys Asp Thr      50              #     55             #     60 Ile Val Asp Ala Ala Cys Ala Val Glu Met Va#l His Ala Ala Ser Leu  65                  # 70                 # 75                  # 80 Ile Phe Asp Asp Leu Pro Cys Met Asp Asp Al#a Gly Leu Arg Arg Gly                  85  #                 90 #                 95 Gln Pro Ala Thr His Val Ala His Gly Glu Se#r Arg Ala Val Leu Gly             100       #           105      #           110 Gly Ile Ala Leu Ile Thr Glu Ala Met Ala Le#u Leu Ala Gly Ala Arg         115           #       120          #       125 Gly Ala Ser Gly Thr Val Arg Ala Gln Leu Va#l Arg Ile Leu Ser Arg     130               #   135              #   140 Ser Leu Gly Pro Gln Gly Leu Cys Ala Gly Gl#n Asp Leu Asp Leu His 145                 1 #50                 1#55                 1 #60 Ala Ala Lys Asn Gly Ala Gly Val Glu Gln Gl#u Gln Asp Leu Lys Thr                 165   #               170  #               175 Gly Val Leu Phe Ile Ala Gly Leu Glu Met Le#u Ala Val Ile Lys Glu             180       #           185      #           190 Phe Asp Ala Glu Glu Gln Thr Gln Met Ile As#p Phe Gly Arg Gln Leu         195           #       200          #       205 Gly Arg Val Phe Gln Ser Tyr Asp Asp Leu Le#u Asp Val Val Gly Asp     210               #   215              #   220 Gln Ala Ala Leu Gly Lys Asp Thr Gly Arg As#p Ala Ala Ala Pro Gly 225                 2 #30                 2#35                 2 #40 Pro Arg Arg Gly Leu Leu Ala Val Ser Asp Le#u Gln Asn Val Ser Arg                 245   #               250  #               255 His Tyr Glu Ala Ser Arg Ala Gln Leu Asp Al#a Met Leu Arg Ser Lys             260       #           265      #           270 Arg Leu Gln Ala Pro Glu Ile Ala Ala Leu Le#u Glu Arg Val Leu Pro         275           #       280          #       285 Tyr Ala Ala Arg Ala     290 <210> SEQ ID NO 38<211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of Artificial #Sequence: Primer #7 <400> SEQUENCE: 38cctggatgac gtgctggaat attcc           #                  #               25 <210> SEQ ID NO 39 <211> LENGTH: 20 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: Primer #8<400> SEQUENCE: 39 caaggcccag atcgcaggcg             #                  #                   # 20 <210> SEQ ID NO 40 <211> LENGTH: 391<212> TYPE: PRT <213> ORGANISM: Flavobacterium sp. R1534<400> SEQUENCE: 40 Met Asp Pro Ile Val Ile Thr Gly Ala Met Ar#g Thr Pro Met Gly Ala   1               5  #                 10 #                 15 Phe Gln Gly Asp Leu Ala Ala Met Asp Ala Pr#o Thr Leu Gly Ala Asp              20      #             25     #             30 Ala Ile Arg Ala Ala Leu Asn Gly Leu Ser Pr#o Asp Met Val Asp Glu          35          #         40         #         45 Val Leu Met Gly Cys Val Leu Ala Ala Gly Gl#n Gly Gln Ala Pro Ala      50              #     55             #     60 Arg Gln Ala Ala Leu Gly Ala Gly Leu Pro Le#u Ser Thr Gly Thr Thr  65                  # 70                 # 75                  # 80 Thr Ile Asn Glu Met Cys Gly Ser Gly Met Ly#s Ala Ala Met Leu Gly                  85  #                 90 #                 95 His Asp Leu Ile Ala Ala Gly Ser Ala Gly Il#e Val Val Ala Gly Gly             100       #           105      #           110 Met Glu Ser Met Ser Asn Ala Pro Tyr Leu Le#u Pro Lys Ala Arg Ser         115           #       120          #       125 Gly Met Arg Met Gly His Asp Arg Val Leu As#p His Met Phe Leu Asp     130               #   135              #   140 Gly Leu Glu Asp Ala Tyr Asp Lys Gly Arg Le#u Met Gly Thr Phe Ala 145                 1 #50                 1#55                 1 #60 Glu Asp Cys Ala Gly Asp His Gly Phe Thr Ar#g Glu Ala Gln Asp Asp                 165   #               170  #               175 Tyr Ala Leu Thr Ser Leu Ala Arg Ala Gln As#p Ala Ile Ala Ser Gly             180       #           185      #           190 Ala Phe Ala Ala Glu Ile Ala Pro Val Thr Va#l Thr Ala Arg Lys Val         195           #       200          #       205 Gln Thr Thr Val Asp Thr Asp Glu Met Pro Gl#y Lys Ala Arg Pro Glu     210               #   215              #   220 Lys Ile Pro His Leu Lys Pro Ala Phe Arg As#p Gly Gly Thr Val Thr 225                 2 #30                 2#35                 2 #40 Ala Ala Asn Ser Ser Ser Ile Ser Asp Gly Al#a Ala Ala Leu Val Met                 245   #               250  #               255 Met Arg Gln Ser Gln Ala Glu Lys Leu Gly Le#u Thr Pro Ile Ala Arg             260       #           265      #           270 Ile Ile Gly His Ala Thr His Ala Asp Arg Pr#o Gly Leu Phe Pro Thr         275           #       280          #       285 Ala Pro Ile Gly Ala Met Arg Lys Leu Leu As#p Arg Thr Asp Thr Arg     290               #   295              #   300 Leu Gly Asp Tyr Asp Leu Phe Glu Val Asn Gl#u Ala Phe Ala Val Val 305                 3 #10                 3#15                 3 #20 Ala Met Ile Ala Met Lys Glu Leu Gly Leu Pr#o His Asp Ala Thr Asn                 325   #               330  #               335 Ile Asn Gly Gly Ala Cys Ala Leu Gly His Pr#o Ile Gly Ala Ser Gly             340       #           345      #           350 Ala Arg Ile Met Val Thr Leu Leu Asn Ala Me#t Ala Ala Arg Gly Ala         355           #       360          #       365 Thr Arg Gly Ala Ala Ser Val Cys Ile Gly Gl#y Gly Glu Ala Thr Ala     370               #   375              #   380 Ile Ala Leu Glu Arg Leu Ser 385                 3 #90<210> SEQ ID NO 41 <211> LENGTH: 388 <212> TYPE: PRT<213> ORGANISM: Flavobacterium sp. R1534 <400> SEQUENCE: 41Asp Pro Arg Leu Ala Val Arg Asp Gln Gln Pr #o Pro Leu Arg Ile Gly  1               5  #                 10  #                 15Gln His His Pro His Glu Pro Gln Arg Thr Th #r Gln Arg Ala Pro Gln             20      #             25      #             30Ile Gly Arg Val Gln His Gly Met Arg His Hi #s Arg Glu Gly Pro Arg         35          #         40          #         45Arg His Gly Ala Arg Ala His Ser Glu Glu Le #u Ala Ala Cys Pro Leu     50              #     55              #     60Arg Lys Val Ala Pro Asp Arg Ala Val Phe Ar #g Cys Ser Asp Gly Pro 65                  # 70                  # 75                  # 80Asp Ala Arg Gly Pro Ala Leu Pro Arg Arg Hi #s Gln Arg Ile Ala His                 85  #                 90  #                 95Glu Pro Phe Arg Asp Asp Val Leu Ile His Gl #y Pro Ser Leu Gln Asn            100       #           105       #           110Arg Ser Pro Ile Leu Ser Arg Asp Gly Ile Va #l Cys Asn Ala Pro Arg        115           #       120           #       125Ala Arg Met Ala Arg Arg Ile Lys Gly Gly Ar #g Asp Met Glu Ile Glu    130               #   135               #   140Gly Arg Val Phe Val Val Thr Gly Ala Ala Se #r Gly Leu Gly Ala Ala145                 1 #50                 1 #55                 1 #60Ser Ala Arg Met Leu Ala Gln Gly Gly Ala Ly #s Val Val Leu Ala Asp                165   #               170   #               175Leu Ala Glu Pro Lys Asp Ala Pro Glu Gly Al #a Val His Ala Ala Cys            180       #           185       #           190Asp Val Thr Asp Ala Thr Ala Ala Gln Thr Al #a Ile Ala Leu Ala Thr        195           #       200           #       205Asp Arg Phe Gly Arg Leu Asp Gly Leu Val As #n Cys Ala Gly Ile Ala    210               #   215               #   220Pro Ala Glu Arg Met Leu Gly Arg Asp Gly Pr #o His Gly Leu Asp Ser225                 2 #30                 2 #35                 2 #40Phe Ala Arg Ala Val Thr Ile Asn Leu Ile Gl #y Ser Phe Asn Met Ala                245   #               250   #               255Arg Leu Ala Ala Glu Ala Met Ala Arg Asn Gl #u Pro Val Arg Gly Glu            260       #           265       #           270Arg Gly Val Ile Val Asn Thr Ala Ser Ile Al #a Ala Gln Asp Gly Gln        275           #       280           #       285Ile Gly Gln Val Ala Tyr Ala Ala Ser Lys Al #a Gly Val Ala Gly Met    290               #   295               #   300Thr Leu Pro Met Ala Arg Asp Leu Ala Arg Hi #s Gly Ile Arg Val Met305                 3 #10                 3 #15                 3 #20Thr Ile Ala Pro Gly Ile Phe Arg Thr Pro Me #t Leu Glu Gly Leu Pro                325   #               330   #               335Gln Asp Val Gln Asp Ser Leu Gly Ala Ala Va #l Pro Phe Pro Ser Arg            340       #           345       #           350Leu Gly Glu Pro Ser Glu Tyr Ala Ala Leu Le #u His His Ile Ile Ala        355           #       360           #       365Asn Pro Met Leu Asn Gly Glu Val Ile Arg Le #u Asp Gly Ala Leu Arg    370               #   375               #   380 Met Ala Pro Lys 385<210> SEQ ID NO 42 <211> LENGTH: 182 <212> TYPE: PRT<213> ORGANISM: Flavobacterium sp. R1534 <400> SEQUENCE: 42Met Thr Gly Thr Arg Met Arg Arg Val Ser Ar #g Ile Ser Ala Pro Ser  1               5  #                 10  #                 15Ser Pro Ile Leu Pro Met Trp Pro Ser Lys Al #a Ala Ala Leu Leu Ala             20      #             25      #             30Val Leu Met Pro Ala Ala Ala Ala Ala Val Gl #u Cys Ala Pro Gly Ser         35          #         40          #         45Leu Val Val Asp Thr Gly Ala Glu Thr Leu Gl #y Phe Arg Val Glu Val     50              #     55              #     60Ala Asp Ser Pro Glu Glu Arg Ala Gln Gly Le #u Met Phe Arg Lys Glu 65                  # 70                  # 75                  # 80Leu Pro Ala Gly Thr Gly Met Leu Phe Ile Ty #r Glu Ser Pro Gln Pro                 85  #                 90  #                 95Val Ser Phe Trp Met Arg Asn Thr Leu Ile Pr #o Leu Asp Met Val Phe            100       #           105       #           110Ala Asp Glu Thr Gly Val Ile Arg His Ile Hi #s Arg Asn Ala Arg Pro        115           #       120           #       125Leu Asp Glu Thr Pro Ile Pro Gly Ala Ala Va #l Gly Asp Pro Asp Pro    130               #   135               #   140Asp Arg Leu Phe Val Leu Glu Ile Ala Gly Gl #y Glu Ala Asp Arg Leu145                 1 #50                 1 #55                 1 #60Gly Leu Lys Pro Gly Gln Pro Met Ala His Pr #o Gly Met Gly Asp Asn                165   #               170   #               175Ala Val Leu Ala Cys Asp             180 <210> SEQ ID NO 43<211> LENGTH: 22 <212> TYPE: DNA<213> ORGANISM: Flavobacterium sp. R1534 <400> SEQUENCE: 43acgaaggcac cgatgacgcc ca            #                  #                 22 <210> SEQ ID NO 44 <211> LENGTH: 25 <212> TYPE: DNA<213> ORGANISM: Flavobacterium sp. R1534 <400> SEQUENCE: 44cggacctggc cgtcgcatga ccatc           #                  #               25 <210> SEQ ID NO 45 <211> LENGTH: 23 <212> TYPE: DNA<213> ORGANISM: Flavobacterium sp. R1534 <400> SEQUENCE: 45cggatcgcaa tacatgagcc atg            #                  #                23 <210> SEQ ID NO 46 <211> LENGTH: 24 <212> TYPE: DNA<213> ORGANISM: Flavobacterium sp. R1534 <400> SEQUENCE: 46ctgcaggaga gagcatgagt tccg           #                  #                24 <210> SEQ ID NO 47 <211> LENGTH: 23 <212> TYPE: DNA<213> ORGANISM: Flavobacterium sp. R1534 <400> SEQUENCE: 47gcaaggggcc ggcatgagca ctt            #                  #                23 <210> SEQ ID NO 48 <211> LENGTH: 21 <212> TYPE: RNA<213> ORGANISM: Flavobacterium sp. <400> SEQUENCE: 48aaaggagggu uucauaugag c            #                  #                   #21 <210> SEQ ID NO 49 <211> LENGTH: 21<212> TYPE: RNA <213> ORGANISM: Flavobacterium sp. <400> SEQUENCE: 49aaaggaggac acgugaugag c            #                  #                   #21 <210> SEQ ID NO 50 <211> LENGTH: 22<212> TYPE: RNA <213> ORGANISM: Flavobacterium sp. <400> SEQUENCE: 50aaaggaggca auugagauga gu            #                  #                 22 <210> SEQ ID NO 51 <211> LENGTH: 22 <212> TYPE: RNA<213> ORGANISM: Flavobacterium sp. <400> SEQUENCE: 51aaaggaggau ccaaucauga cc            #                  #                 22 <210> SEQ ID NO 52 <211> LENGTH: 21 <212> TYPE: RNA<213> ORGANISM: Flavobacterium sp. <400> SEQUENCE: 52aaaggagggu uucuuaugac g            #                  #                   #21 <210> SEQ ID NO 53 <211> LENGTH: 15<212> TYPE: RNA <213> ORGANISM: Bacillus subtilis <400> SEQUENCE: 53ucuuuccucc acuag               #                   #                  #    15 <210> SEQ ID NO 54 <211> LENGTH: 13 <212> TYPE: RNA<213> ORGANISM: Escherichia coli <400> SEQUENCE: 54auuccuccac uag               #                   #                  #      13 <210> SEQ ID NO 55 <211> LENGTH: 32 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: Primer      crtW15 <400> SEQUENCE: 55tatatctaga catatgtccg gtcgtaaacc gg        #                  #          32 <210> SEQ ID NO 56 <211> LENGTH: 40 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: Primer      crtW26 <400> SEQUENCE: 56tatagaattc cacgtgtcaa gcacgaccac cggttttacg      #                  #    40 <210> SEQ ID NO 57 <211> LENGTH: 17 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: Primet      crtW100 <400> SEQUENCE: 57 caygaygcma tgcaygg             #                   #                   #   17 <210> SEQ ID NO 58<211> LENGTH: 17 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of Artificial #Sequence: Primer       crtW101 <400> SEQUENCE: 58caygaygcka tgcaygg              #                   #                  #   17 <210> SEQ ID NO 59 <211> LENGTH: 17 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: Primer      crtW105 <400> SEQUENCE: 59 agrtgrtgyt crtgrtg             #                   #                   #   17 <210> SEQ ID NO 60<211> LENGTH: 17 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of Artificial #Sequence: Primer       crtW106 <400> SEQUENCE: 60agrtgrtgyt cccartg              #                   #                  #   17 <210> SEQ ID NO 61 <211> LENGTH: 31 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: Primer      crtW107 <400> SEQUENCE: 61atcatatgag cgcacatgcc ctgcccaagg c         #                  #          31 <210> SEQ ID NO 62 <211> LENGTH: 33 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: Primer      crtW108 <400> SEQUENCE: 62atctcgagtc acgtgcgctc ctgcgcctcg gcc        #                  #         33 <210> SEQ ID NO 63 <211> LENGTH: 33 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: Primer      crtW113 <400> SEQUENCE: 63atatacatat ggtgtccccc ttggtgcggg tgc        #                  #         33 <210> SEQ ID NO 64 <211> LENGTH: 35 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: Primer      crtW114 <400> SEQUENCE: 64tatggatccg acgcgttccc ggaccgccac aatgc        #                  #       35 <210> SEQ ID NO 65 <211> LENGTH: 43 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: Primer      AmpR1 <400> SEQUENCE: 65tatatcggcc gactagtaag cttcaaaaag gatcttcacc tag     #                  # 43 <210> SEQ ID NO 66 <211> LENGTH: 30 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: Primer      AmpR2 <400> SEQUENCE: 66 atatgaattc aataatattg aaaaaggaag         #                   #           30

What is claimed is:
 1. A process for the preparation of astaxanthin andadonixanthin wherein the process comprises culturing a recombinant cellcontaining farnesyl pyrophosphate and isopentyl pyrophosphate underculture conditions sufficient for the expression of enzymes whichcatalyze the conversion of the farnesyl pyrophosphate and isopentylpyrophosphate to astaxanthin and adonixanthin, the recombinant cellbeing a host cell transformed by an expression vector comprising aregulatory sequence and a polynucleotide containing the following DNAsequences which encode the enzymes: a) a DNA sequence which encodes thegeranylgeranyl pyrophosphate (GGPP) synthase of Flavobacterium sp. R1534(crtE) (SEQ ID NO: 2) or a DNA sequence that hybridizes to acomplementary strand of SEQ ID NO: 1 under the following conditions:hybridization in 7% sodium dodecyl sulfate (SDS), 1% bovine serumalbumin (BSA), 0.5 M Na₂HPO₄, pH 7.2, at 65° C., washing twice for 5minutes each in 2×SSC, 1% SDS, at room temperature, followed by twoadditional washes for 15 minutes each in 0.1% SSC, 0.1% SDS, at 650° C.,wherein the hybrid DNA encodes a polypeptide having geranylgeranylpyrophosphate (GGPP) synthase activity, b) a DNA sequence which encodesthe prephytoene synthase of Flavobacterium sp. R1534 (crtB) (SEQ ID NO:3) or a DNA sequence that hybridizes to SEQ ID NO: 1 under the followingconditions: hybridization in 7% sodium dodecyl sulfate (SDS), 1% bovineserum albumin (BSA), 0.5 M Na₂HPO₄, pH 7.2, at 65° C., washing twice for5 minutes each in 2×SSC, 1% SDS, at room temperature, followed by twoadditional washes for 15 minutes each in 0.1% SSC, 0.1% SDS, at 65° C.,wherein the hybrid DNA encodes a polypeptide having prephytoene synthaseactivity, c) a DNA sequence which encodes the phytoene desaturase ofFlavobacterium sp. R1534 (crtl) (SEQ ID NO: 4) or a DNA sequence thathybridizes to SEQ ID NO: 1 under the following conditions: hybridizationin 7% sodium dodecyl sulfate (SDS), 1% bovine serum albumin (BSA), 0.5 MNa₂HPO₄, pH 7.2, at 65° C., washing twice for 5 minutes each in 2×SSC,1% SDS, at room temperature, followed by two additional washes for 15minutes each in 0.1% SSC, 0.1% SDS, at 65° C., wherein the hybrid DNAencodes a polypeptide having phytoene desaturase activity, d) a DNAsequence which encodes the lycopene cyclase of Flavobacterium sp. R1534(crtY) (SEQ ID NO: 5) or a DNA sequence that hybridizes to SEQ ID NO: 1under the following conditions: hybridization in 7% sodium dodecylsulfate (SDS), 1% bovine serum albumin (BSA). 0.5 M Na₂HPO₄, pH 7.2, at65° C., washing twice for 5 minutes each in 2×SSC, 1% SDS, at roomtemperature, followed by two additional washes for 15 minutes each in0.1% SSC, 0.1% SDS, at 65° C., wherein the hybrid DNA encodes apolypeptide having lycopene cyclase activity, e) a DNA sequence whichencodes the β-carotene β4-oxygenase of Alcaligenes PC-1 (crtW) (SEQ IDNO: 29) or a DNA sequence that hybridizes to a complementary strand ofSEQ ID NO: 28 under the following conditions: hybridization in 7% sodiumdodecyl sulfate (SDS), 1% bovine serum albumin (BSA), 0.5 M Na₂HPO₄, pH7.2, at 65° C., washing twice for 5 minutes each in 2×SSC, 1% SDS, atroom temperature, followed by two additional washes for 15 minutes eachin 0.1% SSC, 0.1% SDS, at 65° C.,, wherein the hybrid DNA encodes apolypeptide having β-carotene β-oxygenase activity, and f) a DNAsequence which encodes the β-carotene hydroxylase of microorganism E-396(crtZ_(E396)) (SEQ ID NO: 34) or a DNA sequence that hybridizes to acomplementary strand of SEQ ID NO: 33 under the following conditions:hybridization in 7% sodium dodecyl sulfate (SDS), 1% bovine serumalbumin (BSA), 0.5 M Na₂HPO₄. pH 7.2, at 65° C., washing twice for 5minutes each in 2×SSC, 1% SDS, at room temperature, followed by twoadditional washes for 15 minutes each in 0.1% SSC, 0.1% SDS, at 65° C.,wherein the hybrid DNA encodes a polypeptide having β-carotenehydroxylase activity and isolating the astaxanthin and adonixanthin fromsuch cells or the culture medium.
 2. A process according to claim 1wherein the DNA sequences are: (a) the DNA sequence which encodes theGGPP synthase of Flavobacterium sp. R1534 (crtE) (SEQ ID NO: 2), (b) theDNA sequence which encodes the prephytoene synthase of Flavobacteriumsp. R1534 (crtB) (SEQ ID NO: 3), (c) the DNA sequence which encodes thephytoene desaturase of Flavobacterium sp. R1534 (crtl) (SEQ ID NO: 4),(d) the DNA sequence which encodes the lycopene cyclase ofFlavobacterium sp. R1534 (crtY) (SEQ ID NO: 5), (e) the DNA sequencewhich encodes the β-carotene β4-oxygenase of Alcaligenes PC-1 (crtW)(SEQ ID NO: 29), and (f) the DNA sequence which encodes the β-carotenehydroxylase of microorganism E-396 (crtZ_(E396)) (SEQ ID NO: 34).
 3. Theprocess of claim 2, wherein: (a) the DNA sequence encoding the GGPPsynthase comprises nucleotides 2521-3408 of SEQ ID NO: 1, (b) the DNAsequence encoding the prephytoene synthase comprises the complement ofnucleotides 3405-4316 of SEQ ID NO: 1, (c) the DNA sequence encoding thephytoene desaturase comprises the complement of nucleotides 4313-5797 ofSEQ ID NO: 1, (d) the DNA sequence encoding the lycopene cyclasecomprises the complement of nucleotides 5794-6942 of SEQ ID NO: 1, (e)the DNA sequence encoding the β-carotene β4-oxygenase comprises thesequence of SEQ ID NO: 28, and (f) the DNA sequence encoding theβ-carotene hydroxylase comprises the sequence of SEQ ID NO:
 33. 4. Aprocess for the preparation of astaxanthin and adonixanthin wherein theprocess comprises culturing a recombinant cell containing farnesylpyrophosphate and isopentyl pyrophosphate under culture conditionssufficient for the expression of enzymes which catalyze the conversionof the farnesyl pyrophosphate and isopentyl pyrophosphate to astaxanthinand adonixanthin, the recombinant cell being a host cell transformed byan expression vector comprising a regulatory sequence and apolynucleotide containing the following DNA sequences which encode theenzymes: (a) a DNA sequence which encodes the geranylgeranylpyrophosphate (GGPP) synthase of Flavobacterium sp. R1534 (crtE) (SEQ IDNO: 2) or a DNA sequence that hybridizes to a complementary strand ofSEQ ID NO: 1 under the following conditions: hybridization in 7% sodiumdodecyl sulfate (SDS), 1% bovine serum albumin (BSA), 0.5 M Na₂HPO₄, pH7.2, at 65° C., washing twice for 5 minutes each in 2×SSC, 1% SDS, atroom temperature, followed by two additional washes for 15 minutes eachin 0.1% SSC, 0.1% SDS, at 65° C., wherein the hybrid DNA encodes apolypeptide having geranylgeranyl pyrophosphate (GGPP) synthaseactivity, (b) a DNA sequence which encodes the prephytoene synthase ofFlavobacterium sp. R1534 (crtB) (SEQ ID NO: 3) or a DNA sequence thathybridizes to SEQ ID NO: 1 under the following conditions: hybridizationin 7% sodium dodecyl sulfate (SDS), 1% bovine serum albumin (BSA), 0.5 MNa₂HPO₄. pH 7.2. at 65° C., washing twice for 5 minutes each in 2×SSC,1% SDS, at room temperature, followed by two additional washes for 15minutes each in 0.1% SSC, 0.1% SDS, at 65° C., wherein the hybrid DNAencodes a polypeptide having prephytoene synthase activity, (c) a DNAsequence which encodes the phytoene desaturase of Flavobacterium sp.R1534 (crtl) (SEQ ID NO: 4) or a DNA sequence that hybridizes to SEQ IDNO: 1 under the following conditions: hybridization in 7% sodium dodecylsulfate (SDS), 1% bovine serum albumin (BSA), 0.5 M Na₂HPO₄, pH 7.2, at65° C., washing twice for 5 minutes each in 2×SSC, 1% SDS, at roomtemperature, followed by two additional washes for 15 minutes each in0.1% SSC, 0.1% SDS, at 65° C., wherein the hybrid DNA encodes apolypeptide having phytoene desaturase activity, (d) a DNA sequencewhich encodes the lycopene cyclase of Flavobacterium sp. R1534 (crtY)(SEQ ID NO: 5) or a DNA sequence that hybridizes to SEQ ID NO: 1 underthe following conditions: hybridization in 7% sodium dodecyl sulfate(SDS), 1% bovine serum albumin (BSA), 0.5 M Na₂HPO₄, pH 7.2, at 65° C.,washing twice for 5 minutes each in 2×SSC, 1% SDS, at room temperature,followed by two additional washes for 15 minutes each in 0.1% SSC, 0.1%SDS, at 65° C., wherein the hybrid DNA encodes a polypeptide havinglycopene cyclase activity, (e) a DNA sequence which encodes theβ-carotene β4-oxygenase of microorganism E-396 (crtW_(E396)) (SEQ ID NO:32) or a DNA sequence that hybridizes to a complementary strand of SEQID NO: 31 under the following conditions: hybridization in 7% sodiumdodecyl sulfate (SDS), 1% bovine serum albumin (BSA), 0.5 M Na₂HPO₄, pH7.2, at 65° C., washing twice for 5 minutes each in 2×SSC, 1% SDS, atroom temperature, followed by two additional washes for 15 minutes eachin 0.1% SSC, 0.1% SDS, at 65° C., wherein the hybrid DNA encodes apolypeptide having β-carotene β4-oxygenase activity, and (f) a DNAsequence which encodes the β-carotene hydroxylase of microorganism E-396(crtZ_(E396)) (SEQ ID NO: 34) or a DNA sequence that hybridizes to acomplementary strand of SEQ ID NO: 33 under the following conditions:hybridization in 7% sodium dodecyl sulfate (SDS), 1% bovine serumalbumin (BSA), 0.5 M Na₂HPO₄, pH 7.2, at 65° C., washing twice for 5minutes each in 2×SSC, 1% SDS, at room temperature, followed by twoadditional washes for 15 minutes each in 0.1% SSC, 0.1% SDS, at 65°C.,wherein the hybrid DNA encodes a polypeptide having β-carotenehydroxylase activity; and isolating the astaxanthin and adonixanthinfrom such cells or the culture medium.
 5. A process according to claim 4wherein the DNA sequences are: (a) the DNA sequence which encodes theGGPP synthase of Flavobacterium sp. R1534 (crtE) (SEQ ID NO: 2), (b) theDNA sequence which encodes the prephytoene synthase of Flavobacteriumsp. R1534 (crtB) (SEQ ID NO: 3), (c) the DNA sequence which encodes thephytoene desaturase of Flavobacterium sp. R1534 (crtl) (SEQ ID NO: 4),(d) the DNA sequence which encodes the lycopene cyclase ofFlavobacterium sp. R1534 (crtY) (SEQ ID NO: 5), (e) the DNA sequencewhich encodes the β-carotene β4-oxygenase of microorganism E-396(crtW_(E396)) (SEQ ID NO: 32), and (f) the DNA sequence which encodesthe β-carotene hydroxylase of microorganism E-396 (crtZ_(E396)) (SEQ IDNO: 34).
 6. The process of claim 5 wherein: (a) the DNA sequenceencoding the GGPP synthase comprises nucleotides 2521-3408 of SEQ ID NO:1, (b) the DNA sequence encoding the prephytoene synthase comprises thecomplement of nucleotides 3405-4316 of SEQ ID NO: 1, (c) the DNAsequence encoding the phytoene desaturase comprises the complement ofnucleotides 4313-5797 of SEQ ID NO: 1, (d) the DNA sequence encoding thelycopene cyclase comprises the complement of nucleotides 5794-6942 ofSEQ ID NO: 1, (e) the DNA sequence encoding the β-carotene β4-oxygenasecomprises the sequence of SEQ ID NO: 31, and (f) the DNA sequenceencoding the β-carotene hydroxylase comprises the sequence of SEQ ID NO:33.